JP6975704B2 - Multicolor detector - Google Patents

Multicolor detector Download PDF

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JP6975704B2
JP6975704B2 JP2018237484A JP2018237484A JP6975704B2 JP 6975704 B2 JP6975704 B2 JP 6975704B2 JP 2018237484 A JP2018237484 A JP 2018237484A JP 2018237484 A JP2018237484 A JP 2018237484A JP 6975704 B2 JP6975704 B2 JP 6975704B2
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light emitting
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隆 穴沢
智 高橋
基博 山崎
佳孝 児玉
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Hitachi High Tech Corp
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本発明は，複数のキャピラリ又はマイクロチップ内部に設けられた複数のチャンネルにレーザビーム等の光を照射し，キャピラリ又はチャンネルの内部に存在する物質が出射する蛍光，燐光，散乱光等を高感度に検出する多色検出装置に関する。 The present invention irradiates a plurality of channels provided inside a plurality of capillaries or microchips with light such as a laser beam, and has high sensitivity to fluorescence, phosphorescence, scattered light, etc. emitted by substances existing inside the capillaries or channels. The present invention relates to a multicolor detection device for detecting light.

分離媒体を充填した複数のキャピラリ（外径１００〜４００μｍ，内径２５〜１００μｍのガラス毛細管）による電気泳動分析を並列処理することによって個々のキャピラリで異なるＤＮＡサンプルの塩基配列解読を一括して行うキャピラリアレイＤＮＡシーケンサが広く利用されている。この機構について次に説明する。市販のキャピラリは，柔軟性を持たせるため，外表面にポリイミドの被覆膜を形成している。各キャピラリの電気泳動路長が一定の部分，例えばキャピラリの試料注入端から３０ｃｍの距離の位置近傍を，被覆膜を除去した状態で同一平面上に揃えて並べ，レーザビームを上記のキャピラリ配列平面に沿って側面方向から照射することで，複数のキャピラリを同時に照射する。以降，本明細書では，上記のキャピラリ配列平面を，単に，配列平面と呼ぶことがある。上記の各キャピラリ内部を電気泳動する蛍光標識ＤＮＡは，レーザビームを通過する際，レーザ照射による励起を受けて蛍光を発光する。ここで，ＤＮＡは，Ａ，Ｃ，Ｇ，Ｔの塩基種に応じて４色の蛍光体に染め分けられている。その結果，各キャピラリのレーザ照射位置は発光点となり，複数の発光点が間隔ｐで直線上に並ぶ。以降，これを発光点アレイと呼ぶ。発光点の数（キャピラリの本数）をｎとすると，発光点アレイの全幅Ｗは，Ｗ＝ｐ＊（ｎ−１）である。例えば，ｐ＝０．３６ｍｍ，ｎ＝２４のとき，Ｗ＝８．２８ｍｍである。蛍光検出装置は，発光点アレイからの各発光を分光しながら一括検出する。この装置構成は，特許文献１の図３に示されている。 By parallel processing electrophoretic analysis with multiple capillaries (glass capillaries with an outer diameter of 100 to 400 μm and an inner diameter of 25 to 100 μm) filled with a separation medium, the base sequence of different DNA samples can be decoded collectively for each capillary. Array DNA sequencers are widely used. This mechanism will be described below. Commercially available capillaries have a polyimide coating on the outer surface to give them flexibility. The part where the electrophoresis path length of each capillary is constant, for example, the vicinity of the position at a distance of 30 cm from the sample injection end of the capillary is aligned on the same plane with the covering film removed, and the laser beam is arranged in the above-mentioned capillary arrangement. By irradiating from the side along the plane, multiple capillaries are irradiated at the same time. Hereinafter, in the present specification, the above capillary array plane may be simply referred to as an array plane. When the fluorescently labeled DNA electrophoresed inside each of the above capillaries passes through a laser beam, it receives excitation by laser irradiation and emits fluorescence. Here, the DNA is dyed into four-color phosphors according to the base species of A, C, G, and T. As a result, the laser irradiation position of each capillary becomes a light emitting point, and a plurality of light emitting points are arranged on a straight line at an interval p. Hereinafter, this is referred to as a light emitting point array. Assuming that the number of light emitting points (the number of capillaries) is n, the total width W of the light emitting point array is W = p * (n-1). For example, when p = 0.36 mm and n = 24, W = 8.28 mm. The fluorescence detector collectively detects each emission from the emission point array while spectroscopically. This device configuration is shown in FIG. 3 of Patent Document 1.

まず，共通集光レンズによって各発光を平行光束化する。以降，「共通」という表現は，複数（ｎ個）の発光点について１個の光学素子を用いる（ｎ：１の対応）という意味で用いる。反対に，「個別」という表現は，１つの発光点について１個の光学素子を用いる（１：１の対応）という意味で用いる。ここで，共通集光レンズの焦点距離をｆ，有効径をＤ１とすると，Ｗ＜ｆ，Ｗ＜Ｄ１である。例えば，ｆ＝５０ｍｍ，Ｄ１＝３６ｍｍである。次に，平行光束をロングパスフィルタに通してレーザビームの波長をカットし，さらに共通透過型回折格子を透過させて各キャピラリの長軸方向，すなわち発光点アレイの配列方向及び共通集光レンズの光軸の両者に直交する方向に波長分散させる。ここで，共通透過型回折格子の有効径をＤＧとすると，検出効率を低下させないためには，Ｄ１≦ＤＧである必要がある。例えば，ＤＧ＝５０ｍｍである。続いて，共通結像レンズで各平行光束を２次元センサ上に結像させる。ここで，共通結像レンズの有効径をＤ２とすると，検出効率を低下させないためには，Ｄ１≦Ｄ２である必要がある。例えば，Ｄ２＝３６ｍｍである。以上により，発光点アレイからの各発光の波長分散スペクトルを一括して取得できる。最後に，各波長分散スペクトルの時間変化を分析することによって４色の蛍光強度の時間変化を求め，塩基種の順番，すなわち塩基配列を決定する。 First, each emission is made into a parallel luminous flux by a common condenser lens. Hereinafter, the expression "common" is used to mean that one optical element is used for a plurality of (n) light emitting points (correspondence of n: 1). On the contrary, the expression "individual" is used to mean that one optical element is used for one light emitting point (1: 1 correspondence). Here, assuming that the focal length of the common condenser lens is f and the effective diameter is D1, W <f and W <D1. For example, f = 50 mm and D1 = 36 mm. Next, the parallel light beam is passed through a long-pass filter to cut the wavelength of the laser beam, and the common transmission type diffraction grating is passed through to pass the parallel light beam in the long axis direction of each capillary, that is, the arrangement direction of the emission point array and the light of the common condenser lens. Wavelength is dispersed in the direction orthogonal to both axes. Here, assuming that the effective diameter of the common transmission type diffraction grating is DG, it is necessary that D1 ≦ DG in order not to reduce the detection efficiency. For example, DG = 50 mm. Subsequently, each parallel luminous flux is imaged on the two-dimensional sensor with a common imaging lens. Here, assuming that the effective diameter of the common imaging lens is D2, it is necessary that D1 ≦ D2 in order not to reduce the detection efficiency. For example, D2 = 36 mm. As described above, the wavelength dispersion spectrum of each emission from the emission point array can be collectively acquired. Finally, the time change of the fluorescence intensity of the four colors is obtained by analyzing the time change of each wavelength dispersion spectrum, and the order of the base species, that is, the base sequence is determined.

４色の蛍光を同時に検出する他の手段が，非特許文献１の図２に示されている。まず，１個の発光領域からの発光を１個の集光レンズ（ここでは，対物レンズ）によって平行光束化する。ここで，発光領域の全幅をＷ，対物レンズの焦点距離をｆ，有効径をＤ１とすると，Ｗ＜ｆ，Ｗ＜Ｄ１である。用いられている対物レンズは，オリンパスのＵＰＬＳＡＰＯ ６０× Ｗであり，Ｗ＝０．４４ｍｍ，ｆ＝３ｍｍ，Ｄ１＝２０ｍｍである。次に，平行光束を１組の３種類のダイクロイックミラーによって４色の４つの平行光束に分割させる。続いて，各平行光束を１組の４個の結像レンズで４つの２次元センサ上にそれぞれ結像させる。ここで，各結像レンズの有効径をＤ２とすると，検出効率を低下させないためには，Ｄ１≦Ｄ２である必要がある。以上により，発光領域の４色の４分割像を一括して取得できる。 Another means of simultaneously detecting the fluorescence of four colors is shown in FIG. 2 of Non-Patent Document 1. First, the light emitted from one light emitting region is converted into a parallel luminous flux by one condenser lens (here, an objective lens). Here, assuming that the entire width of the light emitting region is W, the focal length of the objective lens is f, and the effective diameter is D1, then W <f and W <D1. The objective lens used is Olympus UPLSAPO 60 × W, W = 0.44 mm, f = 3 mm, D1 = 20 mm. Next, the parallel light flux is divided into four parallel light fluxes of four colors by a set of three types of dichroic mirrors. Subsequently, each parallel luminous flux is imaged on four two-dimensional sensors with a set of four imaging lenses. Here, assuming that the effective diameter of each imaging lens is D2, it is necessary that D1 ≦ D2 in order not to reduce the detection efficiency. From the above, it is possible to collectively acquire a 4-split image of 4 colors in the light emitting region.

一方，発光点アレイからの発光を同時に検出する他の手段が，特許文献２の図１に示されている。まず，個別集光レンズアレイによって発光点アレイからの各発光を平行光束化する。ここで，発光点の間隔をｐ，発光点の数をｎとすると，発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）であり，各集光レンズの有効径をＤ１とすると，Ｄ１＜Ｗである。また，Ｄ１＜ｐとすることによって，各集光レンズが直線上に並んだ個別集光レンズアレイとすることができる。次に，各平行光束を個別センサアレイのそれぞれの個別センサに入射させる。以上により，発光点アレイからの発光強度を一括して取得できる。 On the other hand, another means for simultaneously detecting light emission from the light emission point array is shown in FIG. 1 of Patent Document 2. First, each emission from the emission point array is converted into a parallel luminous flux by an individual condenser lens array. Here, assuming that the distance between the light emitting points is p and the number of light emitting points is n, the total width of the light emitting point array is W = p * (n-1), and if the effective diameter of each condenser lens is D1, then D1. <W. Further, by setting D1 <p, it is possible to form an individual condenser lens array in which each condenser lens is arranged in a straight line. Next, each parallel luminous flux is incident on each individual sensor in the individual sensor array. From the above, the emission intensity from the emission point array can be collectively acquired.

特開２００７−１７１２１４号公報Japanese Unexamined Patent Publication No. 2007-172141 特開２０１１−５９０９５号公報Japanese Unexamined Patent Publication No. 2011-59095

Rev Sci Instrum., 2011 Feb;82(2):023701.Rev Sci Instrum., 2011 Feb; 82 (2): 023701.

特許文献１の蛍光検出装置は，各発光点からの発光の集光効率（共通集光レンズによる集光効率），検出効率（集光効率，ロングパスフィルタの透過率，回折格子の回折効率等を踏まえた，センサによる蛍光検出に寄与する発光のトータルの利用効率）が高く，また，回折格子による分光精度も高い。しかしながら，２つの共通レンズ（カメラレンズを利用）を含み，Ｗ＜ｆ，Ｗ＜Ｄ１≦Ｄ２の関係があることから，Ｗ一定とすると，装置の全体サイズが非常に大きく，装置の製造コストが高いことが課題である。例えば，ｆ＝５０ｍｍ，Ｄ１＝３６ｍｍ，Ｄ２＝３６ｍｍの場合，蛍光検出装置の全体サイズは，直径１００ｍｍ，高さ２００ｍｍの円柱の体積（１．６×１０６ｍｍ３）よりも大きくなる。本明細書では，蛍光検出装置の全体サイズを，発光点から結像点までの光学系の占有体積で表現し，センサ自体の占有体積は含めないことにする。また，Ｗ≪ｆ，Ｗ≪Ｄ１とすることはできないため（実現するためには巨大なカメラレンズが必要），光軸付近の発光点（発光点アレイの中央付近に位置する発光点）の検出効率と比較して，光軸から離れた発光点（発光点アレイの端付近に位置する発光点）の検出効率が低下し，発光点毎に感度にばらつきが生じる課題がある。 The fluorescence detection device of Patent Document 1 determines the focusing efficiency of light emitted from each light emitting point (condensing efficiency by a common condensing lens), detection efficiency (condensing efficiency, transmission rate of a long pass filter, diffraction efficiency of a diffraction grating, etc.). Based on this, the total utilization efficiency of light emission that contributes to fluorescence detection by the sensor) is high, and the spectral accuracy by the diffraction grating is also high. However, since it includes two common lenses (using a camera lens) and has a relationship of W <f and W <D1 ≦ D2, if W is constant, the overall size of the device is very large and the manufacturing cost of the device is high. The challenge is that it is expensive. For example, when f = 50 mm, D1 = 36 mm, and D2 = 36 mm, the overall size of the fluorescence detection device is larger than the volume of a cylinder (1.6 × 10 6 mm 3) having a diameter of 100 mm and a height of 200 mm. In the present specification, the overall size of the fluorescence detection device is expressed by the occupied volume of the optical system from the light emitting point to the imaging point, and the occupied volume of the sensor itself is not included. In addition, since W << f and W << D1 cannot be set (a huge camera lens is required to realize it), detection of a light emitting point near the optical axis (light emitting point located near the center of the light emitting point array) is detected. Compared to the efficiency, there is a problem that the detection efficiency of the light emitting point (the light emitting point located near the end of the light emitting point array) far from the optical axis is lowered, and the sensitivity varies from one light emitting point to the other.

しかし，これらの課題を解決すること，すなわち，発光点アレイからの４色の発光を同時に識別しながら検出する装置を小型化及び低コスト化し，各発光の感度ばらつきを低減することはこれまで行われてこなかった。蛍光検出装置を小型化できれば，キャピラリアレイＤＮＡシーケンサを小さな領域に設置できたり，持ち運びできるようになったり，あるいは使い勝手が向上したりする。また，蛍光検出装置の部品点数が減ったり，各部品のサイズが小さくなったりすることによって製造コストが低減される。さらに，各発光点の感度ばらつきを低減することにより，各キャピラリで分析されるサンプルの定量的な比較が可能になり，発光点アレイのトータルの感度及びダイナミックレンジを向上させることができる。これらの結果，キャピラリアレイＤＮＡシーケンサはさらに普及し，より一層，世の中に貢献することができる。 However, it has been possible to solve these problems, that is, to reduce the size and cost of a device that simultaneously identifies and detects the emission of four colors from the emission point array, and to reduce the variation in the sensitivity of each emission. I didn't come. If the fluorescence detection device can be miniaturized, the capillary array DNA sequencer can be installed in a small area, can be carried around, or the usability is improved. In addition, the number of parts of the fluorescence detection device is reduced, and the size of each part is reduced, so that the manufacturing cost is reduced. Furthermore, by reducing the sensitivity variation of each emission point, it is possible to quantitatively compare the samples analyzed in each capillary, and it is possible to improve the total sensitivity and dynamic range of the emission point array. As a result, the capillary array DNA sequencer has become more widespread and can contribute to the world even more.

非特許文献１に示される蛍光検出装置を用いて，同様の発光点アレイからの４色発光の同時蛍光検出を行うことができる。ただし，ここで用いられている対物レンズでは，Ｗ＝０．４４ｍｍであるため，例えば，発光点アレイの全幅Ｗ＝８．２８ｍｍの一部しか検出できない。そこで，対物レンズ及び４つの個別結像レンズの代わりに，キャピラリアレイＤＮＡシーケンサと同様に共通集光レンズ及び４つの共通結像レンズを用いる。このとき，３種類のダイクロイックミラーの有効径をＤＭとすると，平行光束に対して４５°傾けて配置するため，検出効率を低下させないためには，√２×Ｄ１≦ＤＭである必要がある。例えばＤＭ＝７１ｍｍである。したがって，４つのカメラを含めなくても，蛍光検出装置の全体サイズは特許文献１の場合よりもさらに大きくなり，それだけ製造コストも高くなる。これに加えて，４つのカメラが占める空間は大きく，そのコストも非常に高い。発光点毎の感度ばらつきの課題もそのまま残る。 Using the fluorescence detection device shown in Non-Patent Document 1, simultaneous fluorescence detection of four-color emission from a similar emission point array can be performed. However, since the objective lens used here has W = 0.44 mm, for example, only a part of the total width W = 8.28 mm of the emission point array can be detected. Therefore, instead of the objective lens and the four individual imaging lenses, a common condenser lens and four common imaging lenses are used as in the capillary array DNA sequencer. At this time, assuming that the effective diameters of the three types of dichroic mirrors are DM, they are arranged at an angle of 45 ° with respect to the parallel luminous flux. Therefore, in order not to reduce the detection efficiency, it is necessary that √2 × D1 ≦ DM. For example, DM = 71 mm. Therefore, even if the four cameras are not included, the overall size of the fluorescence detection device is further larger than that in the case of Patent Document 1, and the manufacturing cost is also increased accordingly. In addition to this, the space occupied by the four cameras is large and the cost is very high. The problem of sensitivity variation for each light emitting point remains as it is.

一方，特許文献２に示される蛍光検出装置を用いることは，Ｄ１＜Ｗのため，装置サイズを小さくできる可能性があるが，１色の蛍光検出のみに対応していることが課題である。そこで，特許文献１に倣い，回折格子による波長分散と組み合わせることを考える。ｎ個の発光点からの発光を，ｎ個の個別集光レンズで平行光束とし，それぞれをｎ個の個別透過型回折格子を透過させて波長分散させ，ｎ個の個別結像レンズでｎ個の１次元又は２次元の個別センサ上に結像させる。すなわち，特許文献１の蛍光検出装置を小型化し，それをｎ個並列に並べた構成である。ここで，Ｄ１＜ｐ，ｐ＝０．３６ｍｍのため，例えば，Ｄ１＝０．３ｍｍとする。透過型回折格子の有効径ＤＧは，Ｄ１≦ＤＧと同時に，隣接する回折格子との立体障害を受けないため，ＤＧ＜ｐとする必要があり，例えば，ＤＧ＝０．３ｍｍとすれば良い。以上の蛍光検出装置は，特許文献１の場合と比較して装置の小型化が可能であるが，微細な光学部品をそれぞれｎ個ずつ作製し，それぞれを所定の位置に配列することは困難であり，それだけ製造コストが高くなる。また，ＤＧ＝０．３ｍｍの透過型回折格子を作製すること自体が困難である。 On the other hand, using the fluorescence detection device shown in Patent Document 2 may reduce the size of the device because D1 <W, but the problem is that it supports only fluorescence detection of one color. Therefore, following Patent Document 1, consider combining it with wavelength dispersion using a diffraction grating. Light emitted from n light emitting points is converted into parallel light rays by n individual condenser lenses, and each is transmitted through n individual transmission type diffraction gratings to disperse the wavelength, and n individual imaging lenses are used to disperse the wavelength. Image is formed on a one-dimensional or two-dimensional individual sensor. That is, the fluorescence detection device of Patent Document 1 is miniaturized, and n of them are arranged in parallel. Here, since D1 <p, p = 0.36 mm, for example, D1 = 0.3 mm. Since the effective diameter DG of the transmission type diffraction grating is not affected by steric hindrance with the adjacent diffraction grating at the same time as D1 ≦ DG, it is necessary to set DG <p. For example, DG = 0.3 mm may be set. The above fluorescence detection device can be downsized as compared with the case of Patent Document 1, but it is difficult to manufacture n fine optical components and arrange each of them at a predetermined position. Yes, the manufacturing cost will be higher. Moreover, it is difficult to fabricate a transmission type diffraction grating with DG = 0.3 mm.

次に，非特許文献２に倣い，３種類のダイクロイックミラーと組み合わせることを考える。ｎ個の発光点からの発光を，ｎ個の個別集光レンズで平行光束とし，ｎ個の平行光束をそれぞれ，ｎ組×３種類の個別ダイクロイックミラーを用いて，ｎ組の４色の４分割平行光とし，ｎ組×４個の個別結像レンズでｎ組×４個の個別センサ上に結像させる。すなわち，非特許文献１の蛍光検出装置を小型化し，それをｎ個並列に並べた構成である。ここで，Ｄ１＜ｐ，ｐ＝０．３６ｍｍのため，例えば，Ｄ１＝０．２５ｍｍとする。各ダイクロイックミラーの有効径ＤＭは，√２×Ｄ１≦ＤＭと同時に，隣接するダイクロイックミラーとの立体障害を受けないため，ＤＭ＜ｐとする必要があり，例えば，ＤＭ＝０．３５ｍｍとすれば良い。以上の蛍光検出装置は，特許文献１の場合と比較して発光点による感度のばらつきは低減されるが，微細な光学部品をぞれぞれｎ個又はｎ組ずつ作製し，それぞれを所定の位置に配列することは困難であり，それだけ製造コストが高くなる。また，ｎ個の４分割像を立体障害を避けながら配置すること自体が困難である。さらに，ＤＭ＝０．３５ｍｍのダイクロイックミラーを作製すること自体も困難である。 Next, following Non-Patent Document 2, consider combining with three types of dichroic mirrors. Emissions from n light emitting points are converted into parallel light rays by n individual condenser lenses, and n parallel light rays are each used by n sets × 3 types of individual dichroic mirrors, and 4 of 4 colors of n sets. The split parallel light is used, and an image is formed on n sets × 4 individual sensors with n sets × 4 individual imaging lenses. That is, the fluorescence detection device of Non-Patent Document 1 is miniaturized, and n of them are arranged in parallel. Here, since D1 <p, p = 0.36 mm, for example, D1 = 0.25 mm. The effective diameter DM of each dichroic mirror must be DM <p because it does not suffer steric hindrance with the adjacent dichroic mirror at the same time as √2 × D1 ≦ DM. For example, if DM = 0.35 mm. good. In the above fluorescence detection device, the variation in sensitivity depending on the light emitting point is reduced as compared with the case of Patent Document 1, but n or n sets of fine optical components are manufactured for each, and each of them is predetermined. It is difficult to arrange them in positions, and the manufacturing cost increases accordingly. In addition, it is difficult to arrange n 4-split images while avoiding steric hindrance. Furthermore, it is also difficult to manufacture a dichroic mirror with DM = 0.35 mm.

以上では，キャピラリアレイＤＮＡシーケンサの蛍光検出装置に適用することを想定して４色蛍光検出について述べたが，課題はキャピラリあるいは４色蛍光検出に限定されるものではなく，任意の発光点アレイからの発光について２色以上の多色発光検出する場合に共通のものである。 In the above, four-color fluorescence detection has been described assuming that it is applied to the fluorescence detection device of a capillary array DNA sequencer, but the problem is not limited to capillary or four-color fluorescence detection, and any emission point array can be used. This is common when detecting multicolor emission of two or more colors.

本発明による多色検出装置は，複数の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に平行光束とする複数の集光レンズが配列した集光レンズアレイと，その平行光束がそれぞれ並列に入射される，共通かつ少なくとも１個の分光素子と，分光素子によって分光された光束がそれぞれ並列に入射される，共通かつ少なくとも１個のセンサと，を有する。 The multicolor detection device according to the present invention is a condensing lens array in which a plurality of condensing lenses are arranged in which light emission from each emission point of the emission point array in which a plurality of emission points are arranged is individually converted into a parallel luminous flux, and a parallel thereof. It has at least one common and at least one spectroscopic element in which light fluxes are incident in parallel, and common and at least one sensor in which light fluxes dispersed by the spectroscopic elements are incident in parallel.

分光素子としては，回折格子，プリズム，あるいはダイクロイックミラーを用いることができる。 As the spectroscopic element, a diffraction grating, a prism, or a dichroic mirror can be used.

また，本発明による多色検出装置は，複数の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に平行光束とする複数の集光レンズが配列した集光レンズアレイと，その平行光束がそれぞれ並列に入射される，共通かつ少なくとも１個のカラーセンサと，を有する。 Further, the multicolor detection device according to the present invention includes a condenser lens array in which a plurality of condenser lenses in which light emission from each emission point of the emission point array in which a plurality of emission points are arranged is individually converted into a parallel luminous flux. It has a common and at least one color sensor, in which the parallel luminous fluxes are incident in parallel.

また，本発明による多色検出装置は，複数の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に平行光束とする複数の集光レンズが配列した集光レンズアレイと，その平行光束がそれぞれ並列に入射される，共通かつ少なくとも１個のセンサとを有し，発光点の有効径の平均をｄ，集光レンズの焦点距離の平均をｆ，集光レンズの有効径の平均をＤ，集光レンズとセンサの光学的な距離の平均をｇとするとき，
ｆ≦−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ
を満足する。 Further, the multicolor detection device according to the present invention includes a condensing lens array in which a plurality of condensing lenses are arranged in which light emitted from each emission point of the emission point array in which a plurality of emission points are arranged is individually converted into a parallel light beam. It has at least one common and at least one sensor in which the parallel light beams are incident in parallel, the average of the effective diameters of the light emitting points is d, the average of the focal lengths of the condenser lens is f, and the effective diameter of the condenser lens. Let D be the average of, and g be the average of the optical distance between the condenser lens and the sensor.
f ≦ -0.20 * (d / D) * g + 2.8 * D
To be satisfied.

また，本発明による多色検出装置は，複数の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に平行光束とする集光レンズが配列した集光レンズアレイと，その平行光束がそれぞれ並列に入射される，共通かつ少なくとも１個のセンサとを有し，発光点の有効径の平均をｄ，発光点の配列間隔の平均をｐ，集光レンズと前記センサの光学的な距離の平均をｇとするとき，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ
を満足する。 Further, the multicolor detection device according to the present invention is a condensing lens array in which light condensing lenses are arranged in which light emission from each light emission point of the light emission point array in which a plurality of light emission points are arranged is individually converted into a parallel light beam, and a parallel thereof. It has at least one common and at least one sensor in which light beams are incident in parallel, the average of the effective diameters of the light emitting points is d, the average of the arrangement intervals of the light emitting points is p, and the optical condenser lens and the sensor are optically. When the average of the distances is g,
f ≧ 0.95 * (d / p) * g
To be satisfied.

また，本発明による多色検出装置は，複数の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に平行光束とする集光レンズが配列した集光レンズアレイと，その平行光束を，それぞれ個別に集光束とする複数の結像レンズが配列した結像レンズアレイと，集光束がそれぞれ並列に入射される，共通かつ少なくとも１個のセンサとを有し，発光点の有効径の平均をｄ，集光レンズの焦点距離の平均をｆ，集光レンズの有効径の平均をＤ，集光レンズと当該集光レンズに対応する結像レンズとの間の光学的な距離の平均をｇとするとき，
ｆ≦−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ
を満足する。 Further, the multicolor detection device according to the present invention is a condensing lens array in which light emitting points from each light emitting point of the light emitting point array in which a plurality of light emitting points are arranged are individually arranged as parallel light sources, and a condensing lens array thereof. It has an imaging lens array in which a plurality of imaging lenses are individually collected and a common and at least one sensor in which the focused light is incident in parallel, and is effective as a light emitting point. The average diameter is d, the average focal distance of the condenser lens is f, the average effective diameter of the condenser lens is D, and the optical distance between the condenser lens and the imaging lens corresponding to the condenser lens. When the average of is g
f ≦ -0.20 * (d / D) * g + 2.8 * D
To be satisfied.

また，本発明による多色検出装置は，複数の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に平行光束とする集光レンズが配列した集光レンズアレイと，その平行光束を，それぞれ個別に集光束とする複数の結像レンズが配列した結像レンズアレイと，集光束がそれぞれ並列に入射される，共通かつ少なくとも１個のセンサとを有し，発光点の有効径の平均をｄ，発光点の配列間隔の平均をｐ，集光レンズの焦点距離の平均をｆ，集光レンズと当該集光レンズに対応する結像レンズとの間の光学的な距離の平均をｇとするとき，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ
を満足する。 Further, the multicolor detection device according to the present invention is a condensing lens array in which light condensing lenses are arranged in which light emission from each light emission point of the light emission point array in which a plurality of light emission points are arranged is individually converted into a parallel light beam, and a parallel lens array thereof. It has an imaging lens array in which a plurality of imaging lenses are individually collected and a common and at least one sensor in which the focused light is incident in parallel, and is effective as a light emitting point. The average of the diameters is d, the average of the arrangement intervals of the light emitting points is p, the average of the focal distances of the condenser lenses is f, and the optical distance between the condenser lens and the imaging lens corresponding to the condenser lens. When the average is g,
f ≧ 0.95 * (d / p) * g
To be satisfied.

また，本発明によるデバイスは，複数のチャンネルの少なくとも一部が同一平面上に配列したチャンネルアレイと，チャンネルアレイの各チャンネルからの発光をそれぞれ個別に平行光束とする集光レンズが配列した集光レンズアレイとが一体化されたものである。 Further, in the device according to the present invention, a channel array in which at least a part of a plurality of channels is arranged on the same plane and a condenser lens in which light emission from each channel of the channel array is individually made into a parallel light beam are arranged. It is integrated with the lens array.

複数のチャンネルは複数のキャピラリの内部であっても良いし，マイクロチップの内部に形成されていても良い。 The plurality of channels may be inside a plurality of capillaries or may be formed inside a microchip.

本発明によると，発光点アレイからの発光の多色検出を行う装置を小型化することができ，これを用いた様々な装置の全体サイズを小型化することができる。したがって，装置を置くスペースを削減でき，装置の持ち運びも可能となり，装置の使い勝手が向上する。また，装置を構成する部品点数が削減され，部品そのものを小型化することによって，製造コストを低減することが可能である。 According to the present invention, it is possible to reduce the size of a device that detects multicolor of light emitted from a light emitting point array, and it is possible to reduce the overall size of various devices using the device. Therefore, the space for placing the device can be reduced, the device can be carried around, and the usability of the device is improved. In addition, the number of parts that make up the device is reduced, and the manufacturing cost can be reduced by downsizing the parts themselves.

上記した以外の、課題、構成及び効果は、以下の実施形態の説明により明らかにされる。 Issues, configurations and effects other than those described above will be clarified by the description of the following embodiments.

発光点アレイからの各発光を個別集光レンズでそれぞれ平行光束化し，センサ領域に入射させて検出する蛍光検出装置の構成例を示す模式図。The schematic diagram which shows the structural example of the fluorescence detection apparatus which makes each light emission from a light emission point array into a parallel light beam by an individual condenser lens, and makes it incident on a sensor area, and detects it. ｆをパラメータとして，ｇと相対検出光量の関係を示した図。The figure which showed the relationship between g and the relative detection light amount with f as a parameter. Ｄをパラメータとして，相対検出光量が５０％以上となるｇとｆの関係を示した図。The figure which showed the relationship between g and f that the relative detection light amount is 50% or more with D as a parameter. ｐをパラメータとして，クロストーク信号強度比が２５％以下となるｇとｆの関係を示した図。The figure which showed the relationship between g and f which a crosstalk signal intensity ratio is 25% or less with p as a parameter. Ｄ及びｐをパラメータとして，相対検出光量が５０％以上かつクロストーク信号強度比が２５％以下となるｇとｆの関係を示した図。The figure which showed the relationship between g and f which the relative detection light amount is 50% or more and the crosstalk signal intensity ratio is 25% or less, using D and p as parameters. キャピラリアレイＤＮＡシーケンサの装置構成例を示す模式図。The schematic diagram which shows the apparatus configuration example of the capillary array DNA sequencer. 発光点アレイからの発光を，個別集光レンズ及び共通波長分散素子により多色検出する装置構成例を示す断面模式図。Schematic diagram of a cross section showing an example of a device configuration for detecting light emitted from a light emitting point array in multiple colors using an individual condensing lens and a common wavelength dispersion element. 発光点アレイからの発光を，個別集光レンズ，共通波長分散素子，及び集光レンズの光軸に垂直に配置されたセンサにより多色検出する装置構成例を示す断面模式図。A schematic cross-sectional view showing an example of a device configuration in which light emitted from a light emitting point array is detected in multiple colors by an individual condensing lens, a common wavelength dispersion element, and a sensor arranged perpendicular to the optical axis of the condensing lens. 発光点アレイからの発光を，個別集光レンズ，共通凹面反射型回折格子，及び集光レンズの光軸に垂直に配置されたセンサにより多色検出する装置構成例を示す断面模式図。A schematic cross-sectional view showing an example of a device configuration in which light emitted from a light emitting point array is detected in multiple colors by an individual condensing lens, a common concave reflection diffraction grating, and a sensor arranged perpendicular to the optical axis of the condensing lens. 発光点アレイからの発光を，個別集光レンズ，共通ダイクロイックミラーセット，及びセンサにより多色検出する装置構成例を示す断面模式図。A schematic cross-sectional view showing an example of a device configuration that detects light emitted from a light emitting point array in multiple colors using an individual condenser lens, a common dichroic mirror set, and a sensor. 発光点アレイからの発光を，個別集光レンズ，共通ダイクロイックミラーセット，及びセンサにより，波長分散の場合と同等に多色検出する装置構成例を示す断面模式図。A schematic cross-sectional view showing an example of a device configuration that detects light emitted from a light emitting point array by using an individual condenser lens, a common dichroic mirror set, and a sensor in the same multicolor as in the case of wavelength dispersion. 発光点アレイからの発光を，個別集光レンズ，及びカラーセンサにより多色検出する装置構成例を示す断面模式図。A schematic cross-sectional view showing an example of a device configuration that detects multicolored light emitted from a light emitting point array by using an individual condenser lens and a color sensor. 複数のキャピラリを配列するＶ溝アレイと，個別集光レンズアレイを一体化したデバイスの構成例を示す断面模式図。Schematic diagram of a cross section showing a configuration example of a device in which a V-groove array in which a plurality of capillaries are arranged and an individual condenser lens array are integrated. 複数のキャピラリにそれぞれ個別集光レンズを接着したデバイスの構成例を示す断面模式図。Schematic diagram of a cross section showing a configuration example of a device in which individual condenser lenses are bonded to a plurality of capillaries. マルチチャンネルを有するマイクロチップと個別集光レンズアレイが一体化したデバイスの構成例を示す断面模式図。Schematic diagram of a cross section showing a configuration example of a device in which a microchip having multiple channels and an individual condenser lens array are integrated. マルチチャンネルを有するマイクロチップと個別集光レンズアレイが一体化したデバイスと個別ＬＥＤ照明による発光点アレイからの発光を，共通ダイクロイックミラーセット，及びセンサにより多色検出する装置構成例を示す断面模式図。Schematic diagram showing a configuration example of a device that integrates a microchip with multiple channels and an individual condenser lens array, and a device that detects light emitted from a light emitting point array by individual LED lighting in multiple colors using a common dichroic mirror set and a sensor. ..

本発明は，発光点アレイからの４色の発光を同時に識別しながら検出する装置を小型化及び低コスト化し，各発光の感度ばらつきを低減する手段を提供する。最初に，本発明を概観する。 The present invention provides a means for reducing the size and cost of an apparatus for simultaneously identifying and detecting four colors of light emitted from a light emitting point array and reducing variations in the sensitivity of each light emission. First, an overview of the present invention will be given.

まず，発光点アレイからの各発光を個別集光レンズアレイによって平行光束化する。本明細書では，平行光束という表現を多用するが，構成する光要素が厳密な意味で互いに平行な光束を必ずしも意味するのではなく，発光点から全方位に出射した発光の光要素の互いになす角度が，集光レンズによって少なくとも減少し，ゼロに近づいている光束を意味する。ここで，発光点の間隔の平均をｐ，発光点の数及び個別集光レンズの数をｎとすると，発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）である。各集光レンズの焦点距離の平均をｆ，有効径の平均をＤ１とすると，Ｄ１＜Ｗである。また，Ｄ１＜ｐとすることによって，各集光レンズが直線上に並んだ集光レンズアレイとすることができる。ｐ＝０．３６ｍｍ，ｎ＝２４，Ｗ＝８．２８ｍｍに対して，例えば，ｆ＝１ｍｍ，Ｄ１＝０．３ｍｍとする。 First, each emission from the emission point array is converted into a parallel luminous flux by an individual condenser lens array. In the present specification, the expression parallel luminous flux is often used, but the constituent optical elements do not necessarily mean the luminous flux parallel to each other in a strict sense, but the optical elements of the emission emitted from the emission point in all directions form each other. It means a luminous flux whose angle is at least reduced by the condenser lens and is approaching zero. Here, assuming that the average of the intervals between the light emitting points is p, the number of light emitting points and the number of individual condenser lenses are n, the total width of the light emitting point array is W = p * (n-1). Assuming that the average focal length of each condenser lens is f and the average effective diameter is D1, then D1 <W. Further, by setting D1 <p, it is possible to form a condenser lens array in which the condenser lenses are arranged in a straight line. For example, f = 1 mm and D1 = 0.3 mm for p = 0.36 mm, n = 24, and W = 8.28 mm.

次に，各平行光束を分光素子，例えば１個の共通透過型回折格子を透過させて波長分散させる。ここで，透過型回折格子の発光点アレイの配列方向の有効径をＤＧ１，透過型回折格子の各キャピラリの長軸方向の有効径をＤＧ２とすると，検出効率を低下させないため，（Ｗ＋Ｄ１）≦ＤＧ１，Ｄ１≦ＤＧ２とする。例えば，ＤＧ１＝１０ｍｍ，ＤＧ２＝１ｍｍとすれば良い。このとき，２４個の互いに分離した平行光束は，１個の共通透過型回折格子の異なる箇所に入射され，それぞれが並列に波長分散を受ける。各平行光束の径はＤ１＝０．３ｍｍであり，これは回折格子の格子定数と比較すると十分に大きいため，各平行光束はいずれも良好な波長分散を受けることができる。また，波長分散の方向は各キャピラリの長軸方向，すなわち，発光点アレイの配列方向及び各集光レンズの光軸の両者に垂直な方向とする。 Next, each parallel luminous flux is transmitted through a spectroscopic element, for example, one common transmission type diffraction grating to disperse the wavelength. Here, if the effective diameter in the arrangement direction of the emission point array of the transmission type diffraction grating is DG1 and the effective diameter in the major axis direction of each capillary of the transmission type diffraction grating is DG2, the detection efficiency is not lowered, so (W + D1) ≦ Let DG1 and D1 ≦ DG2. For example, DG1 = 10 mm and DG2 = 1 mm may be set. At this time, the 24 parallel light fluxes separated from each other are incident on different points of one common transmission type diffraction grating, and each receives wavelength dispersion in parallel. The diameter of each parallel light flux is D1 = 0.3 mm, which is sufficiently large compared to the lattice constant of the diffraction grating, so that each parallel light flux can receive good wavelength dispersion. The direction of wavelength dispersion is the long axis direction of each capillary, that is, the direction perpendicular to both the arrangement direction of the emission point array and the optical axis of each condenser lens.

続いて，波長分散を受けた各平行光束を，ｎ個の個別結像レンズで，１個の共通２次元センサ上に結像させる。各結像レンズの有効径の平均Ｄ２は，検出効率を低下させないため，Ｄ１≦Ｄ２である必要がある。例えば，Ｄ２＝０．３ｍｍである。以上により，発光点アレイからの各発光の４色の蛍光検出を一括して行うことができる。 Subsequently, each parallel luminous flux that has undergone wavelength dispersion is imaged on one common two-dimensional sensor with n individual imaging lenses. The average D2 of the effective diameters of each imaging lens needs to be D1 ≦ D2 so as not to reduce the detection efficiency. For example, D2 = 0.3 mm. As described above, it is possible to collectively detect the fluorescence of four colors of each emission from the emission point array.

以上の蛍光検出装置によれば，装置サイズが特許文献１の場合と比較して小型化されるだけでなく，回折格子及びセンサを複数の発光点について共通化することによって装置構成が簡略化され，実装が容易化される。蛍光検出装置の全体サイズは，直径１０ｍｍ，高さ２０ｍｍの円柱よりも小さくすることが可能である。また，回折格子とセンサを共通化しているにも関わらず，各発光点について蛍光検出光学系及び検出効率は等価であり，感度ばらつきを低減することが可能である。以上は，透過型回折格子を波長分散プリズムに置き換えても，同様に課題を解決することができる。 According to the above fluorescence detection device, not only the device size is reduced as compared with the case of Patent Document 1, but also the device configuration is simplified by sharing the diffraction grating and the sensor for a plurality of light emitting points. ， Easy to implement. The overall size of the fluorescence detector can be smaller than that of a cylinder having a diameter of 10 mm and a height of 20 mm. Moreover, despite the fact that the diffraction grating and the sensor are shared, the fluorescence detection optical system and the detection efficiency are equivalent for each light emitting point, and it is possible to reduce the sensitivity variation. As described above, even if the transmission type diffraction grating is replaced with a wavelength dispersion prism, the problem can be solved in the same manner.

本発明の別の態様を説明する。まず，発光点アレイからの各発光を個別集光レンズアレイによって平行光束化する。ここで，発光点の間隔の平均をｐ，発光点の数及び集光レンズの数をｎとすると，発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）である。各集光レンズの焦点距離の平均をｆ，有効径の平均をＤ１とすると，Ｄ１＜Ｗである。また，Ｄ１＜ｐとすることによって，各集光レンズが直線上に並んだ集光レンズアレイとすることができる。ｐ＝０．３６ｍｍ，ｎ＝２４，Ｗ＝８．２８ｍｍに対して，例えば，ｆ＝１ｍｍ，Ｄ１＝０．３ｍｍとする。 Another aspect of the present invention will be described. First, each emission from the emission point array is converted into a parallel luminous flux by an individual condenser lens array. Here, assuming that the average of the intervals between the light emitting points is p and the number of light emitting points and the number of condensing lenses are n, the total width of the light emitting point array is W = p * (n-1). Assuming that the average focal length of each condenser lens is f and the average effective diameter is D1, then D1 <W. Further, by setting D1 <p, it is possible to form a condenser lens array in which the condenser lenses are arranged in a straight line. For example, f = 1 mm and D1 = 0.3 mm for p = 0.36 mm, n = 24, and W = 8.28 mm.

次に，各平行光束を，１組の３種類の共通ダイクロイックミラー及び１個の全反射ミラーを各キャピラリの長軸方向，すなわち発光点アレイの配列方向及び各集光レンズの光軸の両者に直交する方向に平行に配列することにより，ｎ組の４色の４分割した平行光束とし，これらの平行光束をキャピラリアレイの配列平面に垂直な方向，すなわち各集光レンズの光軸に平行な方向に進行させる。各ダイクロイックミラー及び全反射ミラーの発光点アレイの配列方向の有効径の平均をＤＭ１，これと直交方向の有効径の平均をＤＭ２とすると，検出効率を低下させないため，（Ｗ＋Ｄ１）≦ＤＭ１，√２×Ｄ１≦ＤＭ２とする。例えば，ＤＭ１＝１０ｍｍ，ＤＭ２＝１ｍｍとすれば良い。このとき，２４個の互いに分離した平行光束は，各種類について１個のダイクロイックミラーの異なる箇所に入射され，それぞれが透過光と反射光に２分割される。１個のダイクロイックミラーはどの箇所でも均一な性能が得られるため，各平行光束はいずれも良好な分光を受けることができる。なお，全反射ミラーはダイクロイックミラーで置き換えても良い。 Next, each parallel beam is applied to a set of three types of common dichroic mirrors and one total reflection mirror in the long axis direction of each capillary, that is, in both the arrangement direction of the emission point array and the optical axis of each condenser lens. By arranging them in parallel in the orthogonal directions, n sets of four colors are divided into four parallel light beams, and these parallel light rays are arranged in the direction perpendicular to the array plane of the capillary array, that is, parallel to the optical axis of each condenser lens. Move in the direction. If the average effective diameter in the arrangement direction of the emission point array of each dichroic mirror and total reflection mirror is DM1 and the average effective diameter in the orthogonal direction is DM2, the detection efficiency is not reduced, so (W + D1) ≤ DM1, √ 2 × D1 ≦ DM2. For example, DM1 = 10 mm and DM2 = 1 mm may be set. At this time, the 24 parallel light fluxes separated from each other are incident on different parts of one dichroic mirror for each type, and each is divided into transmitted light and reflected light. Since one dichroic mirror can obtain uniform performance at any location, each parallel luminous flux can receive good spectroscopy. The total reflection mirror may be replaced with a dichroic mirror.

続いて，ｎ組の４色の４分割した平行光束を，結像させずに，１個の共通２次元センサ上に入射させる。回折格子やプリズムによる波長分散によって分光する場合，上述の通り，波長分散された平行光束を結像レンズを用いて結像しなければ所望の分光精度が得られない。これに対して，ダイクロイックミラーによって分光する場合は必ずしもその必要がないため，結像レンズを省くことが可能である。以上により，発光点アレイからの各発光の４色の蛍光検出を一括して行うことができる。 Subsequently, n sets of four-color, four-divided parallel light fluxes are incident on one common two-dimensional sensor without forming an image. In the case of spectroscopy by wavelength dispersion using a diffraction grating or prism, as described above, the desired spectral accuracy cannot be obtained unless the wavelength-dispersed parallel light beams are imaged using an imaging lens. On the other hand, it is not always necessary to perform spectroscopy with a dichroic mirror, so it is possible to omit the imaging lens. As described above, it is possible to collectively detect the fluorescence of four colors of each emission from the emission point array.

以上の蛍光検出装置によれば，装置サイズが特許文献１の場合と比較して小型化されるだけでなく，ダイクロイックミラー及びセンサを複数の発光点について共通化することによって装置構成が簡略化され，実装が容易化される。蛍光検出装置の全体サイズは，直径１０ｍｍ，高さ１０ｍｍの円柱よりも小さくすることが可能である。また，回折格子とセンサを共通化しているにも関わらず，各発光点について蛍光検出系及び検出効率は等価であり，感度ばらつきを低減することが可能である。 According to the above fluorescence detection device, not only the device size is reduced as compared with the case of Patent Document 1, but also the device configuration is simplified by sharing the dichroic mirror and the sensor for a plurality of light emitting points. ， Easy to implement. The overall size of the fluorescence detector can be smaller than that of a cylinder having a diameter of 10 mm and a height of 10 mm. Moreover, despite the fact that the diffraction grating and the sensor are shared, the fluorescence detection system and the detection efficiency are equivalent for each light emitting point, and it is possible to reduce the sensitivity variation.

本発明の更に別の態様を説明する。まず，発光点アレイからの各発光を個別集光レンズアレイによって平行光束化する。ここで，発光点の間隔の平均をｐ，発光点の数及び集光レンズの数をｎとすると，発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）である。各集光レンズの焦点距離の平均をｆ，有効径の平均をＤ１とすると，Ｄ１＜Ｗである。また，Ｄ１＜ｐとすることによって，各集光レンズが直線上に並んだ集光レンズアレイとすることができる。ｐ＝０．３６ｍｍ，ｎ＝２４，Ｗ＝８．２８ｍｍに対して，例えば，ｆ＝１ｍｍ，Ｄ１＝０．３５ｍｍとする。 Yet another aspect of the invention will be described. First, each emission from the emission point array is converted into a parallel luminous flux by an individual condenser lens array. Here, assuming that the average of the intervals between the light emitting points is p and the number of light emitting points and the number of condensing lenses are n, the total width of the light emitting point array is W = p * (n-1). Assuming that the average focal length of each condenser lens is f and the average effective diameter is D1, then D1 <W. Further, by setting D1 <p, it is possible to form a condenser lens array in which the condenser lenses are arranged in a straight line. For example, f = 1 mm and D1 = 0.35 mm for p = 0.36 mm, n = 24, and W = 8.28 mm.

続いて，各平行光束を，結像させずに，１個の単板の共通２次元カラーセンサに入射させる。カラーセンサは，４色をそれぞれ識別する少なくとも４種類の画素が２次元のセンサ面上にそれぞれ多数配列している，あるいは，多数配列する１種類の各画素で２次元のセンサ面に垂直方向（入射光の進行方向）で４色を識別するものである。ここで，各画素の径の平均をＳとすると，Ｓ＜Ｄ１である必要がある。各平行光束の径の平均はＤ１＝０．３５ｍｍである。これに対して，４色を識別する４種類の画素が配列するセンサを用い，Ｓ＝０．０５ｍｍとすると，各平行光束はカラーセンサ上の約４０画素で検出される。このとき，１色を識別する１種類の画素あたり約１０画素で検出されるため，これらを積算することによって色毎のばらつきを低減し，高精度な分光が可能になる。 Subsequently, each parallel light beam is incident on a common two-dimensional color sensor of one single plate without forming an image. In a color sensor, at least four types of pixels that identify each of the four colors are arranged in large numbers on a two-dimensional sensor surface, or one type of pixel in which a large number of pixels are arranged in a direction perpendicular to the two-dimensional sensor surface ( The four colors are identified by the traveling direction of the incident light). Here, assuming that the average diameter of each pixel is S, it is necessary that S <D1. The average diameter of each parallel luminous flux is D1 = 0.35 mm. On the other hand, if a sensor in which four types of pixels that identify four colors are arranged is used and S = 0.05 mm, each parallel light beam is detected by about 40 pixels on the color sensor. At this time, since it is detected with about 10 pixels per one type of pixel that identifies one color, it is possible to reduce the variation for each color and enable highly accurate spectroscopy by integrating these.

これに対して，各平行光束を個別集光レンズアレイで結像し，例えば，結像スポットの径が０．０５ｍｍになったとすると，カラーセンサ上の約１画素でのみ検出されることになり，良好な分光が不可能になる。つまり，この態様では，結像レンズをあえて用いないことが，装置の小型化に寄与するだけでなく，分光精度を向上することにも寄与する。以上により，発光点アレイからの各発光の４色の蛍光検出を一括して行うことができる。 On the other hand, if each parallel light beam is imaged by an individual condensing lens array and the diameter of the imaging spot is, for example, 0.05 mm, it will be detected only by about one pixel on the color sensor. ， Good spectroscopy becomes impossible. That is, in this aspect, not using the imaging lens intentionally contributes not only to the miniaturization of the apparatus but also to the improvement of the spectral accuracy. As described above, it is possible to collectively detect the fluorescence of four colors of each emission from the emission point array.

以上の蛍光検出装置によれば，装置サイズが特許文献１の場合と比較して小型化されるだけでなく，装置構成が極めて簡単である。蛍光検出装置の全体サイズを，直径１０ｍｍ，高さ５ｍｍの円柱よりも小さくすることが可能である。また，各発光点について蛍光検出系及び検出効率は等価であり，感度ばらつきを低減することが可能である。 According to the above fluorescence detection device, not only the device size is reduced as compared with the case of Patent Document 1, but also the device configuration is extremely simple. The overall size of the fluorescence detector can be made smaller than a cylinder having a diameter of 10 mm and a height of 5 mm. In addition, the fluorescence detection system and detection efficiency are equivalent for each emission point, and it is possible to reduce sensitivity variations.

以上では，キャピラリアレイＤＮＡシーケンサの蛍光検出装置に適用することを想定して４色蛍光検出について述べたが，課題の解決手段はキャピラリあるいは４色蛍光検出に限定されるものではなく，任意の発光点アレイからの発光について２色以上の多色発光検出に共通のものである。 In the above, four-color fluorescence detection has been described assuming that it is applied to a fluorescence detection device of a capillary array DNA sequencer, but the means for solving the problem is not limited to capillary or four-color fluorescence detection, and any emission is possible. Light emission from a point array is common to multicolor emission detection of two or more colors.

以下，図面を参照して詳細に説明する。 Hereinafter, a detailed description will be given with reference to the drawings.

本発明が対象とする発光点アレイの各発光点のサイズは小さいとは言え，有限の大きさを有しており，蛍光検出装置を小型化する際には無視できない。図１は，発光点アレイからの各発光を個別集光レンズでそれぞれ平行光束化し，センサ領域に入射させて検出する蛍光検出装置の構成例を示す模式図である。図１は，平均の有効径ｄの発光点１５からの発光を，平均の焦点距離ｆ，平均の有効径Ｄの個別集光レンズ１８で平行光束化し，個別集光レンズ１８から平均の距離ｇに位置する，平均の有効径Ｄのセンサ領域２８に入射させて検出する構成を示している。センサ領域２８の平均の有効径Ｄは，センサの全体サイズを必ずしも示すものではなく，より大きなサイズのセンサの一部が上記発光点１５の検出のために割り当てられた領域と考えることもできる。また，図１には示さないが，各平行光束を個別結像レンズによって再集光，又は結像してからセンサ領域に入射させて検出する蛍光検出装置の場合には，以降の議論では，センサ領域を個別結像レンズに置き換えれば良く，個別集光レンズと個別結像レンズの平均の距離をｇとすれば良い。 Although the size of each light emitting point of the light emitting point array targeted by the present invention is small, it has a finite size and cannot be ignored when the fluorescence detection device is miniaturized. FIG. 1 is a schematic diagram showing a configuration example of a fluorescence detection device in which each light emitted from a light emitting point array is converted into a parallel luminous flux by an individual condensing lens and incident on a sensor region for detection. In FIG. 1, light emitted from a light emitting point 15 having an average effective diameter d is converted into a parallel light beam by an individual condenser lens 18 having an average focal length f and an average effective diameter D, and the average distance g from the individual condenser lens 18 is shown. The configuration is shown in which the lens is incident on the sensor region 28 having an average effective diameter D and is detected. The average effective diameter D of the sensor region 28 does not necessarily indicate the overall size of the sensor, and can be considered as a region in which a part of the sensor having a larger size is allocated for detecting the light emitting point 15. Further, although not shown in FIG. 1, in the case of a fluorescence detection device that detects each parallel light beam by refocusing or forming an image with an individual imaging lens and then incident on the sensor region, in the following discussions, The sensor region may be replaced with an individual imaging lens, and the average distance between the individual condenser lens and the individual imaging lens may be g.

最初に，図１の左側の発光点１５に着目する。発光点１５の中心からの発光２９が集光レンズ１８によって平行光束３０となり，センサ領域２８上でスポット３１を形成し，センサ領域２８とスポット３１は一致する。このとき検出される光量は，Ｄ一定とすると，ｆが小さいほど受光角θ１とともに大きくなる。より正確には，Ｆ＝ｆ／Ｄのとき，検出光量は１／Ｆ２に比例して大きくなる。一方，発光点１５の左端からの発光３２の平行光束３３のスポット３４はセンサ領域２８から右側にずれる。つまり，スポット３１はすべて検出されるが，スポット３４は，スポット３１と重なった比率でのみ検出される。この重なりが大きいほど発光点の全域について検出される光量が大きくなる。そのためには，平行光束３０の光軸と平行光束３３の光軸のなす角θ２が小さければ良く，さらにそのためには，ｄ一定とすると，ｆが大きいほど良い。以上のように，発光点１５の検出光量を大きくするためには，ｆを小さくした方が良い面と，ｆを大きくした方が良い面のトレードオフの関係があるが，どのようなｆが最も良いかの検討はこれまでになされていない。そこで，次に，発光点１５の検出光量を大きくするためのｆ及びｇの条件を解明する。 First, pay attention to the light emitting point 15 on the left side of FIG. The light emitting 29 from the center of the light emitting point 15 becomes a parallel luminous flux 30 by the condenser lens 18, and a spot 31 is formed on the sensor region 28, and the sensor region 28 and the spot 31 coincide with each other. Assuming that D is constant, the amount of light detected at this time increases with the light receiving angle θ1 as f becomes smaller. More precisely, when F = f / D, the amount of detected light increases in proportion to 1 / F 2. On the other hand, the spot 34 of the parallel luminous flux 33 of the light emitting 32 from the left end of the light emitting point 15 shifts to the right from the sensor region 28. That is, all the spots 31 are detected, but the spots 34 are detected only at a ratio overlapping with the spots 31. The larger the overlap, the larger the amount of light detected over the entire light emitting point. For that purpose, it is sufficient that the angle θ2 formed by the optical axis of the parallel luminous flux 30 and the optical axis of the parallel luminous flux 33 is small, and for that purpose, if d is constant, the larger f is, the better. As described above, in order to increase the amount of detected light at the light emitting point 15, there is a trade-off relationship between the aspect where it is better to reduce f and the aspect where it is better to increase f. No consideration has been given to whether it is the best. Therefore, next, the conditions of f and g for increasing the amount of detected light at the light emitting point 15 will be clarified.

検出光量を評価するため，特許文献１の図３に示された蛍光検出装置を基準とする。この蛍光検出装置の典型例では，共通集光レンズの焦点距離はｆ＝５０ｍｍ，有効径はＤ１≧２５ｍｍである。このレンズの明るさはＦ＝ｆ／Ｄ１≦２．０である。そこで，Ｆ０＝２．０の集光レンズを用いた場合に，焦点に位置する無限小サイズの発光点からの発光が，このレンズによって平行光束化され，その光量がすべてロスなくセンサで検出されるとき，その検出光量を基準（１００％）とする。以降では，任意の無限小サイズの発光点についての検出光量を上記基準に対する相対検出光量で評価する。また，平均の有効径ｄの有限サイズの発光点は，多数の無限小サイズの発光点で構成されていると考える。本明細書では，「有限サイズの発光点」は単に「発光点」と呼び，「無限小サイズの発光点」はその都度「無限小サイズの発光点」と呼ぶ。発光点の相対検出光量は，それを構成する多数の無限小サイズの発光点の相対検出光量の平均とする。例えば，上記の例で，集光レンズをＦ＝１．４で置き換えると，集光効率が（Ｆ０／Ｆ）２＝２．０倍になるので，上記無限小サイズの発光点の相対検出光量は２００％となる。ただし，発光点から全方位に発光される全光量は一定とし，発光点の内部の発光密度は空間的に均一であると仮定する。また，本蛍光検出装置の典型例では，発光点アレイの発光点の間隔がｐ＝０．３６ｍｍ，発光点の数がｎ＝２４，発光点アレイの全幅がＷ＝ｐ＊（ｎ−１）＝８．２８ｍｍであり，発光点アレイの中央に位置する発光点はレンズの焦点近傍に位置するため相対検出光量がほぼ１００％になるが，発光点アレイの端に位置する発光点はレンズの焦点から離れるため相対検出光量が減少し，約５０％となる。そこで，本発明では，各発光点の相対検出光量が５０％以上になるようにして，各発光点の多色検出感度が従来と同等以上になるようにすることを目標とする。 In order to evaluate the amount of detected light, the fluorescence detection device shown in FIG. 3 of Patent Document 1 is used as a reference. In a typical example of this fluorescence detection device, the focal length of the common condenser lens is f = 50 mm, and the effective diameter is D1 ≧ 25 mm. The brightness of this lens is F = f / D1 ≦ 2.0. Therefore, when a condensing lens with F 0 = 2.0 is used, the light emitted from the infinitely small size light emitting point located at the focal point is made into a parallel light beam by this lens, and all the light amount is detected by the sensor without loss. When this is done, the amount of detected light is used as a reference (100%). In the following, the amount of detected light for any infinitesimal size emission point will be evaluated by the amount of detected light relative to the above criteria. Further, it is considered that the finite size light emitting point with the average effective diameter d is composed of a large number of infinitesimal size light emitting points. In the present specification, the "finite size light emitting point" is simply referred to as the "light emitting point", and the "infinitesimal size light emitting point" is referred to as the "infinitesimal size light emitting point" each time. The relative detection light amount of the light emitting point is the average of the relative detection light amounts of many infinitesimal size light emitting points constituting the light emitting point. For example, in the above example, if the condensing lens is replaced with F = 1.4, the condensing efficiency becomes (F 0 / F) 2 = 2.0 times, so the relative detection of the infinitesimal size emission point The amount of light is 200%. However, it is assumed that the total amount of light emitted from the emission point in all directions is constant, and the emission density inside the emission point is spatially uniform. Further, in a typical example of this fluorescence detection device, the distance between the light emitting points of the light emitting point array is p = 0.36 mm, the number of light emitting points is n = 24, and the total width of the light emitting point array is W = p * (n-1). = 8.28 mm, and the emission point located in the center of the emission point array is located near the focal point of the lens, so the relative detection light amount is almost 100%, but the emission point located at the end of the emission point array is the lens. Since it is away from the focal point, the relative detection light amount decreases to about 50%. Therefore, it is an object of the present invention to make the relative detection light amount of each light emitting point 50% or more so that the multicolor detection sensitivity of each light emitting point becomes equal to or higher than the conventional one.

図２は，図１に示した構成において，ｆをパラメータとして，ｇと相対検出光量の関係を計算した結果の図である。ここで，発光点１５の平均の有効径はｄ＝０．０５ｍｍとした。また，個別集光レンズ１８の平均の有効径をＤ＝０．５ｍｍとした。レンズの明るさＦ＝ｆ／０．０５を考慮して相対検出光量を計算した。有効径ｄ＝０．０５ｍｍの発光点１５を，０．１μｍ間隔の約５００個の無限小サイズの発光点で構成し，各無限小サイズの発光点について，図１のスポット３１とスポット３４の重なり面積比と同じ考え方によって相対検出光量を計算し，それらの平均により発光点１５の相対検出光量を求めた。その結果，ｆは小さいほど，またｇは小さいほど相対検出光量が大きくなることを初めて見出した。これは，ｆを小さく（Ｆを小さく，θ１を大きく）することによって発光点１５の中心に位置する無限小サイズの発光点の相対検出光量が増大する効果が，ｆを大きく（θ２を小さく）することによって上記の重なり面積比を増大する効果よりも大きいことを示している。また，任意のｆに対して，ｇを小さくすることによって上記の重なり面積比を増大する効果が大きいことを示している。 FIG. 2 is a diagram of the results of calculating the relationship between g and the relative detection light amount with f as a parameter in the configuration shown in FIG. Here, the average effective diameter of the light emitting point 15 is d = 0.05 mm. Further, the average effective diameter of the individual condenser lenses 18 was set to D = 0.5 mm. The relative detection light amount was calculated in consideration of the brightness F = f / 0.05 of the lens. A light emitting point 15 having an effective diameter d = 0.05 mm is composed of about 500 infinitesimal size light emitting points at intervals of 0.1 μm, and for each infinitesimal size light emitting point, the spot 31 and the spot 34 in FIG. The relative detection light amount was calculated by the same concept as the overlapping area ratio, and the relative detection light amount at the emission point 15 was obtained by averaging them. As a result, it was found for the first time that the smaller f and the smaller g, the larger the relative detection light amount. This is because the effect of increasing the relative detection light amount of the infinitesimal light emitting point located at the center of the light emitting point 15 by making f small (F small, θ1 large) makes f large (θ2 small). It is shown that the effect is larger than the above-mentioned effect of increasing the overlapping area ratio. Further, it is shown that the effect of increasing the overlapping area ratio is large by reducing g with respect to any f.

図３は，図２の計算結果を踏まえて，相対検出光量が５０％以上の条件を満たすｇとｆの関係を，横軸ｇ，縦軸ｆのグラフで示したものである。ここでは，Ｄをパラメータとしている。Ｄがいずれの値の場合も，負の傾きを持つ直線より下側の領域のｇとｆであれば相対検出光量が５０％以上となることが分かった。Ｄが大きくなるほど，境界となる直線の縦軸切片が大きくなり，傾きが小さくなるため，条件を満たす領域が大きくなった。詳細に解析した結果，条件を満たす領域は，一般に，
ｆ≦−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ （１）
で表せることが分かった。図２の結果と同様に，ｆとｇはそれぞれ小さいほど，つまり図３の原点に近いほど相対検出光量が大きくなる。しかし，実際には様々な物理的な制約があるため，図３に示す領域の中から，適当なｆとｇを設定するのが良い。 FIG. 3 shows the relationship between g and f satisfying the condition that the relative detection light amount is 50% or more based on the calculation result of FIG. 2 as a graph on the horizontal axis g and the vertical axis f. Here, D is used as a parameter. It was found that, regardless of the value of D, the relative detection light amount is 50% or more if g and f in the region below the straight line having a negative slope. As D becomes larger, the vertical intercept of the straight line as the boundary becomes larger and the slope becomes smaller, so that the region satisfying the condition becomes larger. As a result of detailed analysis, the areas that satisfy the conditions are generally,
f ≦ −0.20 * (d / D) * g + 2.8 * D (1)
It turned out that it can be expressed by. Similar to the result of FIG. 2, the smaller f and g are, that is, the closer to the origin of FIG. 3, the larger the relative detection light amount. However, since there are actually various physical restrictions, it is better to set appropriate f and g from the region shown in FIG.

一方，図１において，左側の発光点１５と同様に，右側の発光点１５の中心からの発光３５は，集光レンズ１８によって平行光束３６とされ，そのスポット３７はセンサ領域２８と一致してロスなく検出される。しかしながら，左側の発光点１５の左端からの発光３２の平行光束３３のスポット３４はセンサ領域から右側にずれ，右側の発光点のセンサ領域に重なる場合があり，右側の発光点の検出におけるクロストークとなる。クロストークは，スポット３７とスポット３４の重なる比率で示される。ここで，ｄ，ｆ，Ｄ，ｇの各パラメータは左側の発光点と右側の発光点で等しく，両発光点の平均の間隔はｐである。また，図１に示さないが，右側の発光点のさらに右側に隣接する発光点の右端からの発光の平行光束のスポットは，同様に，スポット３７と重なり，右側の発光点１５の検出におけるクロストークとなる。各発光点からの発光を良好に検出するためには，クロストークは小さいほど良く，少なくとも信号強度より小さくなければならない。そこで，本発明では，注目する発光点の両隣の発光点からのクロストークは同等に発生するため，それぞれのクロストーク信号強度比を２５％以下に抑えるようにして，各発光点の低クロストーク検出を実現することを目標とする。 On the other hand, in FIG. 1, similarly to the light emitting point 15 on the left side, the light emitting 35 from the center of the light emitting point 15 on the right side is made into a parallel luminous flux 36 by the condenser lens 18, and the spot 37 coincides with the sensor region 28. Detected without loss. However, the spot 34 of the parallel luminous flux 33 of the light emitting 32 from the left end of the light emitting point 15 on the left side may shift to the right side from the sensor area and overlap the sensor area of the light emitting point on the right side, and crosstalk in detecting the light emitting point on the right side. It becomes. Crosstalk is indicated by the overlapping ratio of spot 37 and spot 34. Here, each parameter of d, f, D, and g is equal at the light emitting point on the left side and the light emitting point on the right side, and the average interval between the two light emitting points is p. Further, although not shown in FIG. 1, the spot of the parallel luminous flux of the light emission from the right end of the light emission point adjacent to the right side of the light emission point on the right side also overlaps with the spot 37 and crosses in the detection of the light emission point 15 on the right side. It will be a talk. In order to detect light emission from each light emission point well, the smaller the crosstalk, the better, and it must be at least smaller than the signal strength. Therefore, in the present invention, since crosstalk from the light emitting points on both sides of the light emitting point of interest is generated equally, the crosstalk signal intensity ratio of each is suppressed to 25% or less, and the low crosstalk of each light emitting point is performed. The goal is to achieve detection.

図４は，クロストーク信号強度比が２５％以下の条件を満たすｇとｆの関係を，横軸ｇ，縦軸ｆのグラフで示したものである。ここで，発光点の平均の有効径はｄ＝０．０５ｍｍとした。ここでは，ｐをパラメータとし，Ｄ＝ｐとした。ｐがいずれの値の場合も，原点を通る正の傾きを持つ直線より上側の領域のｇとｆであればクロストーク信号強度比が２５％以下となることが分かった。パラメータｐが大きくなるほど，傾きが小さくなるため，条件を満たす領域が大きくなった。詳細に解析した結果，条件を満たす領域は，一般に，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ （２）
で表せることが分かった。図３の相対検出光量の場合と異なり，ｆは大きいほど，ｇは小さいほどクロストークを低く抑えることができる。つまり，ｆについては，相対検出光量を大きくする場合と，クロストークを小さくする場合でトレードオフの関係になることが分かった。 FIG. 4 shows the relationship between g and f satisfying the condition that the crosstalk signal intensity ratio is 25% or less as a graph on the horizontal axis g and the vertical axis f. Here, the average effective diameter of the light emitting points was set to d = 0.05 mm. Here, p is a parameter and D = p. It was found that the crosstalk signal intensity ratio was 25% or less if g and f in the region above the straight line having a positive slope passing through the origin regardless of the value of p. The larger the parameter p, the smaller the slope, and the larger the area that satisfies the condition. As a result of detailed analysis, the areas that satisfy the conditions are generally,
f ≧ 0.95 * (d / p) * g (2)
It turned out that it can be expressed by. Unlike the case of the relative detection light amount in FIG. 3, the larger f and the smaller g, the lower the crosstalk can be suppressed. In other words, it was found that there is a trade-off relationship between f when the relative detection light amount is increased and when the crosstalk is decreased.

図５（ａ）は，相対検出光量が５０％以上，かつクロストーク信号強度比が２５％以下の条件を満たすｇとｆの関係を，横軸ｇ，縦軸ｆのグラフで示したものである。図５（ｂ）は図５（ａ）の拡大図である。ここで，発光点の平均の有効径はｄ＝０．０５ｍｍであり，Ｄ＝ｐとした。この条件を満たす領域は，言うまでもなく，図３の領域と図４の領域が重なった領域である。パラメータＤ及びｐが大きくなるほど，条件を満たす領域が大きくなった。この条件を満たす領域は，一般に，式（１）かつ式（２）で表すことができる。 FIG. 5A shows the relationship between g and f satisfying the conditions that the relative detection light amount is 50% or more and the crosstalk signal intensity ratio is 25% or less as a graph on the horizontal axis g and the vertical axis f. be. 5 (b) is an enlarged view of FIG. 5 (a). Here, the average effective diameter of the light emitting points is d = 0.05 mm, and D = p. Needless to say, the region satisfying this condition is a region where the region of FIG. 3 and the region of FIG. 4 overlap. The larger the parameters D and p, the larger the region satisfying the condition. The region satisfying this condition can be generally expressed by Eq. (1) and Eq. (2).

以下，本発明の実施例を説明する。
［実施例１］
図６は，キャピラリアレイＤＮＡシーケンサの装置構成例を示す模式図である。図６を用いて分析手順を説明する。まず，複数のキャピラリ１（図６では４本のキャピラリを示す）の試料注入端２を陰極側緩衝液４に浸し，試料溶出端３をポリマブロック９を介して陽極側緩衝液５に浸す。ポンプブロック９のバルブ１４を閉じ，ポンプブロック９に接続されたシリンジ８により内部のポリマ溶液に加圧し，ポリマ溶液を各キャピラリ１の内部に，試料溶出端３から試料注入端２に向かって充填する。次に，バルブ１４を開け，各試料注入端２から異なる試料を注入した後，陰極６と陽極７の間に電源１３により高電圧を印加することにより，キャピラリ電気泳動を開始する。４色の蛍光体で標識されたＤＮＡは試料注入端２から試料溶出端３に向かって電気泳動される。各キャピラリ１の，試料注入端２より一定距離電気泳動された位置（レーザ照射位置１２）は，被覆が除去され，同一平面上に配列されており，レーザ光源１０より発振されたレーザビーム１１が，集光されてから，配列平面の側方より，配列平面に沿って導入され，各キャピラリ１のレーザ照射位置１２が一括照射される。４色の蛍光体で標識されたＤＮＡがレーザ照射位置１２を通過する際に励起され，蛍光を発光する。各発光蛍光は，配列平面に対して垂直方向（図６の紙面に垂直方向）から蛍光検出装置によって検出される。なお，キャピラリの内部はチャンネルを構成している。従って，キャピラリアレイはチャンネルアレイの一種である。 Hereinafter, examples of the present invention will be described.
[Example 1]
FIG. 6 is a schematic diagram showing an example of a device configuration of a capillary array DNA sequencer. The analysis procedure will be described with reference to FIG. First, the sample injection end 2 of the plurality of capillaries 1 (4 capillaries are shown in FIG. 6) is immersed in the cathode side buffer solution 4, and the sample elution end 3 is immersed in the anode side buffer solution 5 via the polymer block 9. The valve 14 of the pump block 9 is closed, the syringe 8 connected to the pump block 9 pressurizes the internal polymer solution, and the polymer solution is filled inside each capillary 1 from the sample elution end 3 toward the sample injection end 2. do. Next, the valve 14 is opened, different samples are injected from each sample injection end 2, and then a high voltage is applied between the cathode 6 and the anode 7 by the power supply 13 to start capillary electrophoresis. The DNA labeled with the four-color phosphor is electrophoresed from the sample injection end 2 toward the sample elution end 3. At the position (laser irradiation position 12) of each capillary 1 electrophoresed for a certain distance from the sample injection end 2, the coating is removed and the laser beam 11 oscillated from the laser light source 10 is arranged on the same plane. After being focused, it is introduced from the side of the array plane along the array plane, and the laser irradiation position 12 of each capillary 1 is collectively irradiated. The DNA labeled with the four-color phosphor is excited as it passes through the laser irradiation position 12, and emits fluorescence. Each emission fluorescence is detected by the fluorescence detection device from the direction perpendicular to the array plane (direction perpendicular to the paper surface of FIG. 6). The inside of the capillary constitutes a channel. Therefore, the capillary array is a kind of channel array.

図７は，発光点アレイからの発光を，個別集光レンズ，及び共通波長分散素子により多色検出する装置構成例を示す断面模式図である。図７（ａ）はレーザ照射位置における各キャピラリの長軸に垂直な断面を示し，図７（ｂ）は任意の一つのキャピラリの長軸に平行な断面を示す。また，図７（ｃ）は２次元センサで検出される画像を示す。 FIG. 7 is a schematic cross-sectional view showing an example of a device configuration for detecting light emitted from a light emitting point array by using an individual condenser lens and a common wavelength dispersion element for multiple colors. FIG. 7 (a) shows a cross section perpendicular to the long axis of each capillary at the laser irradiation position, and FIG. 7 (b) shows a cross section parallel to the long axis of any one capillary. Further, FIG. 7C shows an image detected by the two-dimensional sensor.

図７に示すように，外径０．３６ｍｍ，内径０．０５ｍｍの４本のキャピラリ１が，レーザ照射位置において，間隔ｐ＝１ｍｍで同一平面上に配列し，径０．０５ｍｍに絞ったレーザビーム１１を配列平面側方より照射することで，数ｎ＝４個，有効径ｄ＝０．０５ｍｍの発光点１５が間隔ｐ＝１ｍｍで配列する発光点アレイを得る。４本のキャピラリ１はキャピラリアレイ，すなわちチャンネルアレイを構成する。発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）＝３ｍｍである。焦点距離ｆ＝２ｍｍ，有効径Ｄ＝１ｍｍの４個の集光レンズ１８が間隔ｐ＝１ｍｍで配列する個別集光レンズアレイ１７を，各集光レンズ１８の焦点位置と各発光点１５が一致するように，また各集光レンズ１７の光軸が配列平面と垂直になるように設置し，各発光点１５からの発光１６をそれぞれ集光して平行光束１９とする。次に，各平行光束１９を，配列平面に平行に配置された共通ロングパスフィルタ２０を並列に透過させてレーザ光をカットする。続いて，各平行光束１９を，配列平面に平行に配置された，格子周波数１０００本／ｍｍ（格子定数１μｍ）の１個の共通透過型回折格子２１を透過させて，各キャピラリ１の長軸方向に波長分散させる。透過型回折格子２１の発光点アレイ方向の有効径はＤＧ１＝５ｍｍ，各キャピラリの長軸方向の有効径はＤＧ２＝３ｍｍとする。 As shown in FIG. 7, four capillarys 1 having an outer diameter of 0.36 mm and an inner diameter of 0.05 mm are arranged on the same plane with an interval p = 1 mm at the laser irradiation position, and the laser is narrowed down to a diameter of 0.05 mm. By irradiating the beam 11 from the side of the arrangement plane, a light emitting point array in which several n = 4 light emitting points 15 having an effective diameter d = 0.05 mm are arranged at an interval p = 1 mm is obtained. The four capillaries 1 form a capillary array, that is, a channel array. The total width of the emission point array is W = p * (n-1) = 3 mm. The individual condenser lens array 17 in which four condenser lenses 18 having a focal length f = 2 mm and an effective diameter D = 1 mm are arranged at an interval p = 1 mm, the focal position of each condenser lens 18 and each emission point 15 coincide with each other. In addition, the optical axis of each condensing lens 17 is installed so as to be perpendicular to the arrangement plane, and the emission 16 from each emission point 15 is focused to obtain a parallel light beam 19. Next, each parallel luminous flux 19 is transmitted in parallel by a common long-pass filter 20 arranged parallel to the array plane to cut the laser beam. Subsequently, each parallel light beam 19 is transmitted through one common transmission type diffraction grating 21 having a lattice frequency of 1000 lines / mm (lattice constant 1 μm) arranged parallel to the arrangement plane, and the long axis of each capillary 1 is transmitted. Wavelength dispersion in the direction. The effective diameter of the transmission type diffraction grating 21 in the light emitting point array direction is DG1 = 5 mm, and the effective diameter of each capillary in the major axis direction is DG2 = 3 mm.

このとき，５００ｎｍ，６００ｎｍ，７００ｎｍの発光の１次回折光は，図７（ｂ）に示すように，配列平面の法線に対して，それぞれ３０．０°，３６．９°，４４．４°の方向に進行する。続いて，焦点距離ｆ’＝２ｍｍ，有効径Ｄ’＝１ｍｍの４個の結像レンズ２３が間隔ｐ’＝１ｍｍで配列する個別結像レンズアレイを，各結像レンズ２３の光軸を配列平面の法線に対して３６．９°傾け，６００ｎｍの１次回折光の光軸とそれぞれ一致させ，透過型回折格子２１と近接させて設置し，波長分散された各平行光束２２を等倍に結像する。５００ｎｍ，６００ｎｍ，７００ｎｍの平行光束２２は結像レンズ２３によって，それぞれ集光束２４，２５，２６となる。ここで，６００ｎｍの平行光束２２を基準として，各集光レンズ１８と対応する各結像レンズ２３の間隔（光路長）をｇ＝５ｍｍとする。このとき，ｆ＝２ｍｍに対して，−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ＝２．７５ｍｍとなり，式（１）が満足され，相対検出光量は９６％（＞５０％）となる。また，０．９５＊（ｄ／ｐ）＊ｇ＝０．２４ｍｍとなり，式（２）が満足され，クロストーク信号強度比が０．４％（＜２５％）となる。さらに，結像レンズアレイから２ｍｍ離れた位置に１個の共通２次元ＣＣＤのセンサ面２７を結像レンズアレイと平行に設置し，各発光１６の波長分散像４７を検出する。 At this time, the first-order refracted light of 500 nm, 600 nm, and 700 nm is 30.0 °, 36.9 °, and 44.4 ° with respect to the normal of the array plane, respectively, as shown in FIG. 7 (b). Proceed in the direction of. Subsequently, an individual imaging lens array in which four imaging lenses 23 having a focal length f'= 2 mm and an effective diameter D'= 1 mm are arranged at an interval p'= 1 mm is arranged, and the optical axis of each imaging lens 23 is arranged. Tilt 36.9 ° with respect to the normal line of the plane, align it with the optical axis of the primary diffracted light of 600 nm, install it close to the transmission type diffraction grid 21, and make each wavelength-dispersed parallel light beam 22 equal magnification. Image image. The 500 nm, 600 nm, and 700 nm parallel luminous fluxes 22 have the luminous fluxes 24, 25, and 26, respectively, depending on the imaging lens 23. Here, the distance (optical path length) between each condenser lens 18 and the corresponding imaging lens 23 is set to g = 5 mm with respect to the parallel luminous flux 22 of 600 nm. At this time, for f = 2 mm, −0.20 * (d / D) * g + 2.8 * D = 2.75 mm, the equation (1) is satisfied, and the relative detection light amount is 96% (> 50%). ). Further, 0.95 * (d / p) * g = 0.24 mm, the equation (2) is satisfied, and the crosstalk signal intensity ratio is 0.4% (<25%). Further, a sensor surface 27 of one common two-dimensional CCD is installed in parallel with the imaging lens array at a position 2 mm away from the imaging lens array, and the wavelength dispersion image 47 of each emission 16 is detected.

図７（ｃ）は２次元ＣＣＤで検出される画像５１を示しており，各発光１６の各波長分散像４７が１ｍｍ間隔で配列している。各波長分散像４７には，５００ｎｍ，６００ｎｍ，７００ｎｍの集光束２４，２５，２６の結像スポット４８，４９，５０が含まれる。波長分散の方向と発光点アレイの方向は垂直であるため，各発光の波長分散像は互いに重なることなく，独立に検出される。ＣＣＤの画素サイズを０．０５ｍｍ角とすると，約２０ｎｍ／画素の波長分解能が得られる。また，各発光点１５は等倍で結像されるため，波長分散されない場合の結像サイズは０．０５ｍｍ，すなわち画素サイズと等しい。つまり，結像サイズが波長分解能を低下させることはない。４色検出を行う場合，各ピーク波長は，５００〜７００ｎｍの範囲で，間隔が２０〜３０ｎｍである場合が多い。したがって，各ピーク波長はＣＣＤ上で間隔が１画素以上となり，識別可能である。 FIG. 7C shows an image 51 detected by a two-dimensional CCD, in which each wavelength dispersion image 47 of each emission 16 is arranged at 1 mm intervals. Each wavelength dispersion image 47 includes imaging spots 48, 49, 50 of the luminous fluxes 24, 25, 26 of 500 nm, 600 nm, and 700 nm. Since the direction of the wavelength dispersion and the direction of the emission point array are perpendicular to each other, the wavelength dispersion images of each emission are detected independently without overlapping with each other. Assuming that the pixel size of the CCD is 0.05 mm square, a wavelength resolution of about 20 nm / pixel can be obtained. Further, since each light emitting point 15 is imaged at the same magnification, the image size when the wavelength is not dispersed is 0.05 mm, that is, equal to the pixel size. That is, the imaging size does not reduce the wavelength resolution. When performing four-color detection, each peak wavelength is in the range of 500 to 700 nm, and the interval is often 20 to 30 nm. Therefore, each peak wavelength has an interval of one pixel or more on the CCD and can be identified.

各発光点に対応する波長分散像，すなわち波長分散スペクトルの時間変化を分析することによって，４色の蛍光強度の時間変化を求め，塩基種の順番，すなわち塩基配列を決定する。５００〜７００ｎｍの波長分散像の長さは０．５ｍｍ程度であるため，２次元ＣＣＤのセンサ面のサイズは，発光点アレイ方向に５ｍｍ以上，波長分散方向に１ｍｍ以上あれば十分である。 By analyzing the wavelength dispersion image corresponding to each emission point, that is, the time change of the wavelength dispersion spectrum, the time change of the fluorescence intensity of the four colors is obtained, and the order of the base species, that is, the base sequence is determined. Since the length of the wavelength dispersion image of 500 to 700 nm is about 0.5 mm, it is sufficient that the size of the sensor surface of the two-dimensional CCD is 5 mm or more in the emission point array direction and 1 mm or more in the wavelength dispersion direction.

以上の蛍光検出装置の全体サイズは，キャピラリの長軸方向の幅が５ｍｍ，配列平面と垂直方向の幅が１０ｍｍ，発光点アレイ方向の幅が５ｍｍで規定される直方体の体積（２５０ｍｍ２）よりも小さい。すなわち，特許文献１の場合と比較して，蛍光検出装置の全体サイズを１／６，４００倍に小型化できる。また，用いる光学素子はいずれも微細であるため，大幅な低コスト化が可能である。さらに，本蛍光検出装置による各発光の多色検出感度は高く，かつ均一であり，多色識別精度は高く，クロストークも低い。上記の実施例では発光点の数がｎ＝４であったが，数に限りはなく，数が増えても同様の効果を発揮することができる。透過型の回折格子の代わりに分散プリズムを用いても良い。また，以上では，複数のキャピラリを用いた電気泳動によるＤＮＡシーケンス，４色蛍光検出を対象としていたが，本発明の対象はキャピラリ，ＤＮＡシーケンス，４色蛍光検出のいずれにも限定されるものではなく，複数の発光点からの発光を多色検出する場合のすべてを対象としている。 The overall size of the above fluorescence detection device is based on the volume of a rectangular parallelepiped (250 mm 2 ) defined by the width of the capillary in the long axis direction of 5 mm, the width in the direction perpendicular to the array plane of 10 mm, and the width in the direction of the emission point array of 5 mm. Is also small. That is, the overall size of the fluorescence detection device can be reduced by 1/6,400 times as compared with the case of Patent Document 1. Moreover, since all the optical elements used are minute, it is possible to significantly reduce the cost. Furthermore, the multicolor detection sensitivity of each emission emitted by this fluorescence detection device is high and uniform, the multicolor discrimination accuracy is high, and the crosstalk is low. In the above embodiment, the number of light emitting points was n = 4, but the number is not limited, and the same effect can be exhibited even if the number increases. A dispersion prism may be used instead of the transmission type diffraction grating. Further, in the above, the target is DNA sequence and 4-color fluorescence detection by electrophoresis using a plurality of capillaries, but the subject of the present invention is not limited to any of the capillary, DNA sequence, and 4-color fluorescence detection. It is intended for all cases where multicolor detection of emission from multiple emission points is performed.

以上では，図７（ｂ）に示すように，波長分散に伴い，各平行光束の光路が配列平面の法線から傾いている。このため，２次元ＣＣＤを配列平面に対して傾ける必要があるため，場合により，他の要素と立体障害を生じることがある。図７（ｂ）に示すように，ＣＣＤのセンサ面は，波長分散方向の幅が１ｍｍ以上あれば良く，これが立体障害を引き起こす可能性は低いが，ＣＣＤの回路基板や筐体（いずれも図７に示さず）が立体障害を引き起こす可能性がある。例えば，図７の場合で，ＣＣＤの全体の波長分散方向の幅が２７ｍｍを超えると（センサ面は波長分散方向の幅の中央に位置すると仮定），ＣＣＤとキャピラリの配列平面が衝突する。このような問題を回避するためには，キャピラリの配列平面とＣＣＤのセンサ面を平行に配置するのが良い。 In the above, as shown in FIG. 7 (b), the optical path of each parallel luminous flux is tilted from the normal of the array plane due to the wavelength dispersion. For this reason, it is necessary to tilt the two-dimensional CCD with respect to the array plane, which may cause steric hindrance with other elements in some cases. As shown in FIG. 7B, the sensor surface of the CCD may have a width of 1 mm or more in the wavelength dispersion direction, which is unlikely to cause steric hindrance, but the circuit board and housing of the CCD (both are shown in the figure). (Not shown in 7) can cause steric hindrance. For example, in the case of FIG. 7, if the width of the entire CCD in the wavelength dispersion direction exceeds 27 mm (assuming that the sensor surface is located at the center of the width in the wavelength dispersion direction), the CCD and the array plane of the capillary collide with each other. In order to avoid such a problem, it is preferable to arrange the array plane of the capillary and the sensor plane of the CCD in parallel.

これを実現するため，図８のように低分散プリズム９７を透過型回折格子２１の後段に配置することによって，透過型回折格子２１を透過した各平行光束２２の進行方向と逆向きに屈折させ，低分散プリズム９７を透過した各平行光束２２の進行方向が配列平面と垂直になるようにする。低分散プリズム９７の材質には，分散の小さいガラスを用いる。例えば，ガラス材がＳＫ１６（ｎｄ＝１．６２，νｄ＝６０．３），頂角５０°の低分散プリズムの一辺を，配列平面と平行に，透過型回折格子２１に近接させて配置する。このとき，波長６００ｎｍの平行光束は，低分散プリズム９７に３６．９°の入射角で一辺から入射し，他辺から５０°の出射角で，つまり配列平面と垂直方向に出射する。各集光レンズ１８と対応する各結像レンズ２３の間の光路長はｇ＝５ｍｍのままとし，相対検出光量，クロストーク信号強度比は上記と同じとする。したがって，図７と同等の多色検出性能を有しながら，２次元ＣＣＤのセンサ面２７とキャピラリ１の配列平面を平行にすることができ，２次元ＣＣＤと配列平面の立体障害を回避できる。このような構成は，蛍光検出装置を小型化すればするほど有効である。ここでは，透過型回折格子２１の後段に低分散プリズム９７を配置したが，他の位置に配置しても構わない。波長分散素子として回折格子の代わりに分散プリズムを用いる場合は，分散プリズムと低分散プリズムを組み合わせた直視プリズムとすれば良い。 In order to realize this, by arranging the low dispersion prism 97 in the subsequent stage of the transmission type diffraction grating 21 as shown in FIG. 8, the parallel light beam 22 transmitted through the transmission type diffraction grating 21 is refracted in the direction opposite to the traveling direction. , The traveling direction of each parallel light beam 22 transmitted through the low-dispersion prism 97 is set to be perpendicular to the arrangement plane. Glass with a small dispersion is used as the material of the low dispersion prism 97. For example, one side of a low-dispersion prism having a glass material of SK16 (nd = 1.62, νd = 60.3) and an apex angle of 50 ° is arranged parallel to the array plane and close to the transmission type diffraction grating 21. At this time, the parallel light beam having a wavelength of 600 nm is incident on the low-dispersion prism 97 from one side at an incident angle of 36.9 °, and is emitted from the other side at an emission angle of 50 °, that is, in the direction perpendicular to the array plane. The optical path length between each condensing lens 18 and each imaging lens 23 is left at g = 5 mm, and the relative detection light amount and the crosstalk signal intensity ratio are the same as above. Therefore, the sensor surface 27 of the two-dimensional CCD and the array plane of the capillary 1 can be made parallel to each other while having the same multicolor detection performance as that of FIG. 7, and the three-dimensional obstacle between the two-dimensional CCD and the array plane can be avoided. Such a configuration becomes more effective as the fluorescence detection device becomes smaller. Here, the low-dispersion prism 97 is arranged after the transmission type diffraction grating 21, but it may be arranged at another position. When a dispersion prism is used instead of the diffraction grating as the wavelength dispersion element, a direct-view prism that combines a dispersion prism and a low dispersion prism may be used.

図９は，発光点アレイからの発光を，個別集光レンズ，共通凹面反射型回折格子，及び集光レンズの光軸に垂直に配置されたセンサにより多色検出する装置構成例を示す模式図である。図９（ａ）はレーザ照射位置における各キャピラリの長軸に垂直な断面を示し，図９（ｂ）は任意の一つのキャピラリの長軸に平行な断面を示す。 FIG. 9 is a schematic diagram showing an example of a device configuration in which light emitted from a light emitting point array is detected in multiple colors by an individual condenser lens, a common concave reflection diffraction grating, and a sensor arranged perpendicular to the optical axis of the condenser lens. Is. FIG. 9A shows a cross section perpendicular to the long axis of each capillary at the laser irradiation position, and FIG. 9B shows a cross section parallel to the long axis of any one capillary.

図９に示すように，共通透過型回折格子の代わりに共通凹面反射型回折格子３８を用いれば，回折格子が結像レンズの役割を兼ねるため，個別結像レンズアレイを省くことができ，蛍光検出装置の一層の小型化が可能である。ここでも，２次元センサのセンサ面２７とキャピラリの配列平面を平行になるように配置し，小型化に伴う立体障害を回避することができる。波長分散の方向は，上記と同様に，キャピラリの長軸方向と一致させる。各集光レンズ１８は，焦点距離ｆ＝２ｍｍ，有効径Ｄ＝１ｍｍ，間隔ｐ＝１ｍｍは上記と同じであるが，各集光レンズ１８と凹面反射型回折格子３８の距離はｇ＝２ｍｍ，凹面反射型回折格子３８の焦点距離はｆ’＝４ｍｍとする。このとき，相対検出光量は９８％に向上し，クロストーク信号強度比が０．１％に低減される。しかし，このままでは，結像レンズ（凹面反射型回折格子３８）が共通化されているため，各平行光束１９が同じ位置に結像され各発光１６の波長分散像が２次元センサ上で一致するので，各発光１６を独立に多色検出することができなくなる。ここでは，この課題を解決するため，個別集光レンズアレイ１７の各集光レンズ１８の光軸を平行から互いにずらすことにより，各平行光束１９の結像位置をずらす。 As shown in FIG. 9, if the common concave reflection type diffraction grating 38 is used instead of the common transmission type diffraction grating, the diffraction grating also serves as an imaging lens, so that the individual imaging lens array can be omitted and fluorescence can be obtained. The detection device can be further miniaturized. Here, too, the sensor surface 27 of the two-dimensional sensor and the array plane of the capillary can be arranged so as to be parallel to each other, and steric hindrance due to miniaturization can be avoided. The direction of wavelength dispersion coincides with the long axis direction of the capillary in the same manner as above. The focal length f = 2 mm, effective diameter D = 1 mm, and spacing p = 1 mm of each condenser lens 18 are the same as above, but the distance between each condenser lens 18 and the concave reflection diffractive lattice 38 is g = 2 mm. The focal length of the concave reflection type diffraction grid 38 is f'= 4 mm. At this time, the relative detection light amount is improved to 98%, and the crosstalk signal intensity ratio is reduced to 0.1%. However, as it is, since the imaging lens (concave reflection type diffraction grating 38) is standardized, each parallel light beam 19 is imaged at the same position, and the wavelength dispersion image of each emission 16 matches on the two-dimensional sensor. Therefore, it becomes impossible to independently detect each light emission 16 in multiple colors. Here, in order to solve this problem, the image formation position of each parallel light beam 19 is shifted by shifting the optical axis of each condenser lens 18 of the individual condenser lens array 17 from parallel to each other.

例えば，図９（ａ）に示すように，１点鎖線で示す各集光レンズ１８の光軸と実線で示す配列平面の法線のなす角度を，θ１＝３°，θ２＝１°，θ３＝−１°，θ４＝−３°のように，互いに２°ずつずらし，各平行光束１９が放射状に広がるようにする。ただし，各集光レンズ１８の各焦点位置は各発光点１５からずれないように配置する。このとき，各発光の結像位置は互いに０．１４ｍｍの距離だけ離れ，この距離は各発光の結像サイズ０．１ｍｍ（像倍率が２倍となるため）よりも大きいため，各発光を独立に多色検出することが可能となる。 For example, as shown in FIG. 9A, the angles formed by the optical axis of each condenser lens 18 indicated by the alternate long and short dash line and the normal of the array plane indicated by the solid line are θ 1 = 3 ° and θ 2 = 1 °. , Θ 3 = -1 °, θ 4 = -3 °, and so on, so that each parallel light beam 19 spreads radially. However, each focal position of each condenser lens 18 is arranged so as not to deviate from each emission point 15. At this time, the imaging positions of each emission are separated from each other by a distance of 0.14 mm, and this distance is larger than the imaging size of each emission of 0.1 mm (because the image magnification is doubled), so that each emission is independent. It is possible to detect multiple colors.

図７のように，レーザビーム１１を複数のキャピラリ１の配列平面の側方より照射する場合，各キャピラリの界面におけるレーザ反射によって，各キャピラリのレーザ照射強度が，レーザビーム１１の入射側（図７（ａ）の右側）から出射側（図７（ａ）の左側）に向かって徐々に低下する。したがって，集光レンズ１８以降の蛍光検出装置が各発光点１５について同等の効率を有していたとしても，得られる蛍光検出強度，あるいは感度は，レーザビーム照射の後段のキャピラリ１（図７（ａ）の左側）ほど低くなる場合がある。このような不均一を解消するため，各集光レンズ１８の集光効率を発光点１５毎に変化させることが有効である。例えば，レーザビーム１１の入射側の集光レンズ１８の有効径を小さく，レーザビーム１１の出射側の集光レンズ１８の有効径を大きくすることは有効である。 As shown in FIG. 7, when the laser beam 11 is irradiated from the side of the arrangement plane of the plurality of capillaries 1, the laser irradiation intensity of each capillary is increased by the laser reflection at the interface of each capillary (FIG. 7). It gradually decreases from the right side of 7 (a) to the exit side (left side of FIG. 7 (a)). Therefore, even if the fluorescence detection device after the condenser lens 18 has the same efficiency for each emission point 15, the obtained fluorescence detection intensity or sensitivity is the capillary 1 after the laser beam irradiation (FIG. 7 (FIG. 7). It may be lower as it is on the left side of a). In order to eliminate such non-uniformity, it is effective to change the condensing efficiency of each condensing lens 18 for each light emitting point 15. For example, it is effective to reduce the effective diameter of the condenser lens 18 on the incident side of the laser beam 11 and increase the effective diameter of the condenser lens 18 on the exit side of the laser beam 11.

［実施例２］
図１０は，発光点アレイからの発光を，個別集光レンズ，共通ダイクロイックミラーセット，及びセンサにより多色検出する装置構成例を示す模式図である。図１０（ａ）はレーザ照射位置における各キャピラリの長軸に垂直な断面を示し，図１０（ｂ）は任意の一つのキャピラリの長軸に平行な断面を示す。また，図１０（ｃ）は２次元センサで検出される画像を示す。 [Example 2]
FIG. 10 is a schematic diagram showing an example of a device configuration for detecting multicolor of light emitted from a light emitting point array by using an individual condenser lens, a common dichroic mirror set, and a sensor. FIG. 10A shows a cross section perpendicular to the long axis of each capillary at the laser irradiation position, and FIG. 10B shows a cross section parallel to the long axis of any one capillary. Further, FIG. 10C shows an image detected by the two-dimensional sensor.

図１０に示すように，外径０．３６ｍｍ，内径０．０５ｍｍの４本のキャピラリ１のレーザ照射位置を間隔ｐ＝０．５ｍｍで同一平面上に配列し，径０．０５ｍｍに絞ったレーザビーム１１を配列平面側方より照射することで，数ｎ＝４個，有効径ｄ＝０．０５ｍｍの発光点１５が間隔ｐ＝０．５ｍｍで配列する発光点アレイを得る。ここで，レーザビーム１１の波長は５０５ｎｍ，４色の蛍光（発光極大波長）は，Ａ蛍光（５４０ｎｍ），Ｂ蛍光（５７０ｎｍ），Ｃ蛍光（６００ｎｍ），及びＤ蛍光（６３０ｎｍ）とする。発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）＝１．５ｍｍである。焦点距離ｆ＝１ｍｍ，有効径Ｄ＝０．４ｍｍの４個の集光レンズ１８が間隔ｐ＝０．５ｍｍで配列する個別集光レンズアレイ１７を，各集光レンズ１８の焦点位置と各発光点１５が一致するように，また各集光レンズ１８の光軸が配列平面と垂直になるように設置し，各発光点１５からの発光をそれぞれ集光して平行光束１９とする。 As shown in FIG. 10, laser irradiation positions of four capillarys 1 having an outer diameter of 0.36 mm and an inner diameter of 0.05 mm are arranged on the same plane with an interval of p = 0.5 mm, and the diameter is narrowed down to 0.05 mm. By irradiating the beam 11 from the side of the arrangement plane, a light emitting point array in which several n = 4 light emitting points 15 having an effective diameter d = 0.05 mm are arranged at an interval p = 0.5 mm is obtained. Here, the wavelength of the laser beam 11 is 505 nm, and the fluorescence of four colors (maximum emission wavelength) is A fluorescence (540 nm), B fluorescence (570 nm), C fluorescence (600 nm), and D fluorescence (630 nm). The total width of the emission point array is W = p * (n-1) = 1.5 mm. The individual condenser lens array 17 in which four condenser lenses 18 having a focal length f = 1 mm and an effective diameter D = 0.4 mm are arranged at an interval p = 0.5 mm is arranged with the focal position of each condenser lens 18 and each emission. The points 15 are set so as to coincide with each other and the optical axis of each condensing lens 18 is perpendicular to the arrangement plane, and the light emitted from each light emitting point 15 is focused to form a parallel light beam 19.

次に，各平行光束１９を，光軸方向に直列に配列された１組の共通ダイクロイックミラーセットに並列に入射させる。ダイクロイックミラーセットは，ロングパスフィルタ５６，Ａダイクロイックミラー３９，Ｂダイクロイックミラー４１，Ｃダイクロイックミラー４３，及びＤダイクロイックミラー４５の５点の要素で構成され，各要素はそれぞれ１個ずつであり，それぞれが各発光点について共通かつ並列に用いられる。ロングパスフィルタ５６は，配列平面と平行に，各集光レンズ１８から０．５ｍｍの距離の位置に配置する。各ダイクロイックミラー３９，４１，４３，４５は，キャピラリの長軸と平行に１ｍｍ間隔で配置し，それぞれ法線が配列平面に対して４５°傾くように配置する。また，Ａダイクロイックミラー３９の中心を各集光レンズから１ｍｍの距離（ロングパスフィルタ５６から０．５ｍｍの距離）の位置に配置する。各要素のサイズは，発光点アレイの配列方向の有効径がＤＭ１＝３ｍｍ，これと直交方向の有効径がＤＭ２＝１．４ｍｍ（ロングパスフィルタのみＤＭ２＝１ｍｍ）である。 Next, each parallel luminous flux 19 is incident in parallel on a set of common dichroic mirror sets arranged in series in the optical axis direction. The dichroic mirror set consists of five elements: a long pass filter 56, an A dichroic mirror 39, a B dichroic mirror 41, a C dichroic mirror 43, and a D dichroic mirror 45, and each element is one. It is used in common and in parallel for each light emitting point. The long pass filter 56 is arranged at a distance of 0.5 mm from each condenser lens 18 in parallel with the array plane. The dichroic mirrors 39, 41, 43, 45 are arranged at 1 mm intervals parallel to the major axis of the capillary, and the normals are arranged so as to be tilted by 45 ° with respect to the arrangement plane. Further, the center of the A dichroic mirror 39 is arranged at a position of 1 mm from each condenser lens (distance of 0.5 mm from the long pass filter 56). As for the size of each element, the effective diameter in the arrangement direction of the emission point array is DM1 = 3 mm, and the effective diameter in the orthogonal direction is DM2 = 1.4 mm (DM2 = 1 mm only for the long pass filter).

各平行光束１９を，最初にロングパスフィルタ５６に垂直に並列に入射させ，５２０ｎｍ以下の光をカットし，特にレーザビームの波長である５０５ｎｍを大幅にカットする。ロングパスフィルタ５６を透過した各平行光束を，次にＡダイクロイックミラー３９に４５°で並列に入射させ，５３０〜５５０ｎｍの光を透過させ，５６０ｎｍ以上の光を反射させる。Ａダイクロイックミラー３９の透過光である５３０〜５５０ｎｍの各平行光束をＡ平行光束４０と呼び，主にＡ蛍光（極大発光波長５４０ｎｍ）の検出に用いる。Ａダイクロイックミラー３９の反射光である各平行光束を，次にＢダイクロイックミラー４１に４５°で並列に入射させ，５６０〜５８０ｎｍの光を反射させ，５９０ｎｍ以上の光を透過させる。Ｂダイクロイックミラー４１の反射光である５６０〜５８０ｎｍの各平行光束をＢ平行光束４２と呼び，主にＢ蛍光（極大発光波長５７０ｎｍ）の検出に用いる。 Each parallel luminous flux 19 is first incident in parallel perpendicularly to the long pass filter 56 to cut light of 520 nm or less, and particularly to significantly cut the wavelength of the laser beam of 505 nm. Each parallel light beam transmitted through the long pass filter 56 is then incident on the A dichroic mirror 39 in parallel at 45 ° to transmit light of 530 to 550 nm and reflect light of 560 nm or more. Each parallel light beam of 530 to 550 nm, which is the transmitted light of the A dichroic mirror 39, is called an A parallel light beam 40, and is mainly used for detecting A fluorescence (maximum emission wavelength 540 nm). Each parallel light beam reflected from the A dichroic mirror 39 is then incident on the B dichroic mirror 41 in parallel at 45 ° to reflect light of 560 to 580 nm and transmit light of 590 nm or more. Each parallel light beam of 560 to 580 nm, which is the reflected light of the B dichroic mirror 41, is called a B parallel light beam 42, and is mainly used for detecting B fluorescence (maximum emission wavelength 570 nm).

Ｂダイクロイックミラー４１の透過光である各平行光束を，次にＣダイクロイックミラー４３に４５°で並列に入射させ，５９０〜６１０ｎｍの光を反射させ，６２０ｎｍ以上の光を透過させる。Ｃダイクロイックミラー４３の反射光である５９０〜６１０ｎｍの各平行光束をＣ平行光束４４と呼び，主にＣ蛍光（極大発光波長６００ｎｍ）の検出に用いる。Ｃダイクロイックミラー４３の透過光である各平行光束を，次にＤダイクロイックミラー４５に４５°で並列に入射させ，６２０〜６４０ｎｍの光を反射させ，６５０ｎｍ以上の光を透過させる（図１０に示さず）。Ｄダイクロイックミラー４５の反射光である６２０〜６４０ｎｍの平行光束をＤ平行光束４６と呼び，主にＤ蛍光（極大発光波長６３０ｎｍ）の検出に用いる。４個の各発光点に対応する４組の各平行光束４０，４２，４４，４６はいずれも，配列平面と垂直方向に進行する。なお，Ｄダイクロイックミラー４５は全反射ミラーで置き換えても良い。 Each parallel light beam transmitted from the B dichroic mirror 41 is then incidented in parallel on the C dichroic mirror 43 at 45 ° to reflect light of 590 to 610 nm and transmit light of 620 nm or more. Each parallel light beam of 590 to 610 nm, which is the reflected light of the C dichroic mirror 43, is called a C parallel light beam 44, and is mainly used for detecting C fluorescence (maximum emission wavelength 600 nm). Each parallel light beam that is transmitted light of the C dichroic mirror 43 is then incident on the D dichroic mirror 45 in parallel at 45 ° to reflect light of 620 to 640 nm and transmit light of 650 nm or more (shown in FIG. 10). figure). The parallel light beam of 620 to 640 nm, which is the reflected light of the D dichroic mirror 45, is called a D parallel light beam 46, and is mainly used for detecting D fluorescence (maximum emission wavelength 630 nm). Each of the four sets of parallel luminous fluxes 40, 42, 44, 46 corresponding to each of the four emission points travels in the direction perpendicular to the array plane. The D dichroic mirror 45 may be replaced with a total reflection mirror.

続いて，共通２次元ＣＣＤのセンサ面２７を配列平面と平行に，各集光レンズから２ｍｍの距離（各ダイクロイックミラー３９，４１，４３，４５の中心から１ｍｍの距離）の位置に配置し，４組の各平行光束４０，４２，４４，４６を結像させずにセンサ面２７に並列に入射させる。図１０（ｃ）に示す２次元ＣＣＤで撮像した画像５１上には，平行光束４０，４２，４４，４６に対応するスポット５２，５３，５４，５５がそれぞれ４組，合計１６個のスポットが形成される。各スポットは，径０．４ｍｍ，発光点アレイ方向に０．５ｍｍ間隔，キャピラリ長軸方向に１ｍｍ間隔で格子状に配列され，それぞれが独立に検出される。したがって，２次元ＣＣＤのセンサ面２７のサイズは，発光点アレイ方向に３ｍｍ以上，キャピラリ長軸方向に５ｍｍ以上あれば十分である。このとき，最も光路長の長いＤ平行光束４６を基準とすると，各集光レンズ１８とセンサ面２７の距離はｇ＝５ｍｍである。ｆ＝１ｍｍに対して，−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ＝１ｍｍとなり，式（１）が満足され，相対検出光量は５１％（＞５０％）となる。また，０．９５＊（ｄ／ｐ）＊ｇ＝０．４８ｍｍとなり，式（２）が満足され，クロストーク信号強度比が０．１％（＜２５％）となる。各発光点に対応する４個のスポットの強度の時間変化を分析することによって，４色の蛍光強度の時間変化を求め，塩基種の順番，すなわち塩基配列を決定する。 Subsequently, the sensor surface 27 of the common two-dimensional CCD is placed parallel to the arrangement plane at a distance of 2 mm from each condenser lens (a distance of 1 mm from the center of each dichroic mirror 39, 41, 43, 45). Each of the four sets of parallel light beams 40, 42, 44, 46 is incident on the sensor surface 27 in parallel without forming an image. On the image 51 captured by the two-dimensional CCD shown in FIG. 10 (c), there are four sets of spots 52, 53, 54, 55 corresponding to the parallel luminous fluxes 40, 42, 44, 46, respectively, for a total of 16 spots. It is formed. Each spot is arranged in a grid pattern with a diameter of 0.4 mm, an interval of 0.5 mm in the emission point array direction, and an interval of 1 mm in the major axis direction of the capillary, and each is detected independently. Therefore, it is sufficient that the size of the sensor surface 27 of the two-dimensional CCD is 3 mm or more in the light emitting point array direction and 5 mm or more in the capillary long axis direction. At this time, the distance between each condenser lens 18 and the sensor surface 27 is g = 5 mm, based on the D parallel luminous flux 46 having the longest optical path length. For f = 1 mm, −0.20 * (d / D) * g + 2.8 * D = 1 mm, the equation (1) is satisfied, and the relative detection light amount is 51% (> 50%). Further, 0.95 * (d / p) * g = 0.48 mm, the equation (2) is satisfied, and the crosstalk signal intensity ratio is 0.1% (<25%). By analyzing the time change of the intensity of the four spots corresponding to each emission point, the time change of the fluorescence intensity of the four colors is obtained, and the order of the base species, that is, the base sequence is determined.

以上の蛍光検出装置の全体サイズは，キャピラリの長軸方向の幅が５ｍｍ，配列平面と垂直方向の幅が５ｍｍ，発光点アレイ方向の幅が３ｍｍで規定される直方体の体積（７５ｍｍ２）よりも小さい。すなわち，特許文献１の場合と比較して，蛍光検出装置の全体サイズを１／２１，０００倍に小型化できる。また，用いる光学素子はいずれも微細であるため，大幅な低コスト化が可能である。さらに，本蛍光検出装置による各発光の多色検出感度は高く，かつ均一であり，多色識別精度は高く，クロストークも低い。上記の実施例では発光点の数がｎ＝４であったが，数に限りはなく，数が増えても同様の効果を発揮することができる。ダイクロイックミラーセットを用いて多色検出を行う他の効果は，特許文献１や実施例１で用いる回折格子の場合と比較して，実効的な検出光量が多いことである。回折格子を用いる場合は波長分散に活用できる回折効率は５０％程度であるのに対して，ダイクロイックミラーセットを用いる場合は殆どロスがないため，実効的には，上記の相対検出光量の２倍程度の光量を得ることができる。 The overall size of the above fluorescence detection device is based on the volume of a rectangular parallelepiped (75 mm 2 ) defined by the width of the capillary in the long axis direction of 5 mm, the width in the direction perpendicular to the array plane of 5 mm, and the width in the direction of the emission point array of 3 mm. Is also small. That is, the overall size of the fluorescence detection device can be reduced by 1/21,000 times as compared with the case of Patent Document 1. Moreover, since all the optical elements used are minute, it is possible to significantly reduce the cost. Furthermore, the multicolor detection sensitivity of each emission emitted by this fluorescence detection device is high and uniform, the multicolor discrimination accuracy is high, and the crosstalk is low. In the above embodiment, the number of light emitting points was n = 4, but the number is not limited, and the same effect can be exhibited even if the number increases. Another effect of performing multicolor detection using the dichroic mirror set is that the effective amount of detected light is larger than that of the diffraction grating used in Patent Document 1 and Example 1. When using a diffraction grating, the diffraction efficiency that can be utilized for wavelength dispersion is about 50%, but when using a dichroic mirror set, there is almost no loss, so it is effectively twice the above relative detection light amount. A degree of light can be obtained.

図１１は，発光点アレイからの発光を，個別集光レンズ，共通ダイクロイックミラーセット，及びセンサにより，波長分散の場合と同等に多色検出する装置構成例を示す断面模式図である。 FIG. 11 is a schematic cross-sectional view showing an example of a device configuration that detects light emitted from a light emitting point array by using an individual condenser lens, a common dichroic mirror set, and a sensor in the same manner as in the case of wavelength dispersion.

図１１は，上記の共通ダイクロイックミラーセットを発展させたものである。ダイクロイックミラーセットは，光軸方向に順番に配列されたロングパスフィルタ５６，ダイクロイックミラー５７，５９，６１，６３，６５，６７，６９，７１の９点の要素で構成される。ダイクロイックミラー５７は，５２０〜５４０ｎｍの平行光束５８を透過させ，５４０ｎｍ以上の光を反射させる。ダイクロイックミラー５９は，５４０〜５６０ｎｍの平行光束６０を反射させ，５６０ｎｍ以上の光を透過させる。ダイクロイックミラー６１は，５６０〜５８０ｎｍの平行光束６２を反射させ，５８０ｎｍ以上の光を透過させる。ダイクロイックミラー６３は，５８０〜６００ｎｍの平行光束６４を反射させ，６００ｎｍ以上の光を透過させる。ダイクロイックミラー６５は，６００〜６２０ｎｍの平行光束６６を反射させ，６２０ｎｍ以上の光を透過させる。ダイクロイックミラー６７は，６２０〜６４０ｎｍの平行光束６８を反射させ，６４０ｎｍ以上の光を透過させる。ダイクロイックミラー６９は，６４０〜６６０ｎｍの平行光束７０を反射させ，６６０ｎｍ以上の光を透過させる。ダイクロイックミラー７１は，６６０〜６８０ｎｍの平行光束７２を反射させ，６８０ｎｍ以上の光を透過させる。 FIG. 11 is an extension of the above-mentioned common dichroic mirror set. The dichroic mirror set is composed of nine elements: a long pass filter 56 arranged in order in the optical axis direction, and a dichroic mirror 57, 59, 61, 63, 65, 67, 69, 71. The dichroic mirror 57 transmits a parallel light flux 58 of 520 to 540 nm and reflects light of 540 nm or more. The dichroic mirror 59 reflects a parallel light flux 60 of 540 to 560 nm and transmits light of 560 nm or more. The dichroic mirror 61 reflects a parallel light flux 62 of 560 to 580 nm and transmits light of 580 nm or more. The dichroic mirror 63 reflects a parallel light flux 64 of 580 to 600 nm and transmits light of 600 nm or more. The dichroic mirror 65 reflects a parallel light flux 66 of 600 to 620 nm and transmits light of 620 nm or more. The dichroic mirror 67 reflects a parallel light flux 68 of 620 to 640 nm and transmits light of 640 nm or more. The dichroic mirror 69 reflects a parallel light flux 70 of 640 to 660 nm and transmits light of 660 nm or more. The dichroic mirror 71 reflects a parallel light flux 72 of 660 to 680 nm and transmits light of 680 nm or more.

以上により，１つの発光点１５からの発光がセンサ面２７上に８個のスポットを形成し，これらの強度は５２０〜６８０ｎｍの範囲の２０ｎｍ幅の発光スペクトルを与える。このような構成にすることにより，用いる蛍光体の種類に応じてダイクロイックミラーを設計し直す必要がなくなり，５２０〜６８０ｎｍの範囲の任意の蛍光を高感度，高精度に検出することが可能となる。各発光を分割するスポットの数，分割する波長幅は，上記の実施例に限らず，任意に設定できることは言うまでもない。 As described above, the light emitted from one light emitting point 15 forms eight spots on the sensor surface 27, and these intensities give an emission spectrum having a width of 20 nm in the range of 520 to 680 nm. With such a configuration, it is not necessary to redesign the dichroic mirror according to the type of phosphor used, and it becomes possible to detect arbitrary fluorescence in the range of 520 to 680 nm with high sensitivity and high accuracy. .. Needless to say, the number of spots for dividing each light emission and the wavelength width for dividing are not limited to the above embodiments, and can be arbitrarily set.

［実施例３］
図１２は，発光点アレイからの発光を，個別集光レンズ，及びカラーセンサにより多色検出する装置構成例を示す断面模式図である。図１２（ａ）はレーザ照射位置における各キャピラリの長軸に垂直な断面を示し，図１０（ｂ）は２次元センサで検出される画像を示す。本実施例は，２次元センサ，例えば２次元ＣＣＤにカラーセンサ面７３を用いる例である。 [Example 3]
FIG. 12 is a schematic cross-sectional view showing an example of a device configuration for detecting multicolor of light emitted from a light emitting point array by using an individual condenser lens and a color sensor. FIG. 12 (a) shows a cross section perpendicular to the long axis of each capillary at the laser irradiation position, and FIG. 10 (b) shows an image detected by the two-dimensional sensor. This embodiment is an example in which a color sensor surface 73 is used for a two-dimensional sensor, for example, a two-dimensional CCD.

各発光点１５からロングパスフィルタ５６までは，実施例２の図１０と同じ構成である。図１２（ａ）に示すように，ロングパスフィルタ５６を透過した各平行光束は直接，２次元ＣＣＤのカラーセンサ面７３に入射される。各集光レンズ１８とカラーセンサ面７３の距離はｇ＝１ｍｍとする。このとき，ｆ＝１ｍｍに対して，−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ＝１．１ｍｍとなり，式（１）が満足され，相対検出光量は６１％（＞５０％）となる。また，０．９５＊（ｄ／ｐ）＊ｇ＝０．１０ｍｍとなり，式（２）が満足され，クロストーク信号強度比が０．０％（＜２５％）となる。 Each light emitting point 15 to the long pass filter 56 has the same configuration as that of FIG. 10 of the second embodiment. As shown in FIG. 12A, each parallel light flux transmitted through the long pass filter 56 is directly incident on the color sensor surface 73 of the two-dimensional CCD. The distance between each condenser lens 18 and the color sensor surface 73 is g = 1 mm. At this time, for f = 1 mm, −0.20 * (d / D) * g + 2.8 * D = 1.1 mm, the equation (1) is satisfied, and the relative detection light amount is 61% (> 50%). ). Further, 0.95 * (d / p) * g = 0.10 mm, the equation (2) is satisfied, and the crosstalk signal intensity ratio is 0.0% (<25%).

図１２（ｂ）に示すように，カラーセンサ面７３の像７４上には，各平行光束によるスポット７５が形成される。各スポット７５は，径Ｄ＝０．４ｍｍ，発光点アレイ方向に０．５ｍｍ間隔で配列され，それぞれが独立に検出される。図１２（ｂ）の拡大図に模式的に示すように，カラーセンサ面７３は，主にＡ蛍光（極大発光波長５４０ｎｍ）を検出するＡ画素７６，主にＢ蛍光（極大発光波長５７０ｎｍ）を検出するＢ画素７７，主にＣ蛍光（極大発光波長６００ｎｍ）を検出するＣ画素７８，主にＤ蛍光（極大発光波長６３０ｎｍ）を検出するＤ画素７９の４種類の画素がそれぞれ多数個，規則正しく配列して構成されている。各画素７６，７７，７８，７９のサイズはいずれもＳ＝０．０５ｍｍであり，Ｓ＜Ｄを満たす。このとき，各スポット７５は約８０個の画素で検出され，１種類の画素あたり約２０画素で検出される。このように，各種類の画素について，多数個の画素が各スポット７５を検出することによって，各発光点１５からの発光の多色検出を精度良く行うことができる。例えば，各種類の画素と，スポットの相対位置が変動したとしても問題にならない。あるいは，スポット内の光強度分布が不均一であったとしても，各色を均等に検出できる。 As shown in FIG. 12B, spots 75 due to the parallel luminous fluxes are formed on the image 74 of the color sensor surface 73. Each spot 75 has a diameter D = 0.4 mm and is arranged at intervals of 0.5 mm in the emission point array direction, and each is independently detected. As schematically shown in the enlarged view of FIG. 12B, the color sensor surface 73 mainly has A pixel 76 for detecting A fluorescence (maximum emission wavelength 540 nm) and mainly B fluorescence (maximum emission wavelength 570 nm). There are a large number of four types of pixels, B pixel 77 for detection, C pixel 78 for mainly detecting C fluorescence (maximum emission wavelength 600 nm), and D pixel 79 for mainly detecting D fluorescence (maximum emission wavelength 630 nm), regularly. It is organized in an array. The size of each pixel 76, 77, 78, 79 is S = 0.05 mm, and S <D is satisfied. At this time, each spot 75 is detected by about 80 pixels, and is detected by about 20 pixels per one type of pixel. In this way, for each type of pixel, a large number of pixels detect each spot 75, so that multicolor detection of light emitted from each light emitting point 15 can be performed with high accuracy. For example, it does not matter if the relative position of each type of pixel and the spot fluctuates. Alternatively, even if the light intensity distribution in the spot is non-uniform, each color can be detected evenly.

以上の蛍光検出装置の全体サイズは，キャピラリの長軸方向の幅が３ｍｍ，配列平面と垂直方向の幅が２ｍｍ，発光点アレイ方向の幅が３ｍｍで規定される直方体の体積（１８ｍｍ２）よりも小さい。すなわち，特許文献１の場合と比較して，蛍光検出装置の全体サイズを１／８９，０００倍に小型化できる。また，用いる光学素子はいずれも微細であるため，大幅な低コスト化が可能である。さらに，本蛍光検出装置による各発光の多色検出感度は高く，かつ均一であり，多色識別精度は高く，クロストークも低い。上記の実施例では発光点の数がｎ＝４であったが，数に限りはなく，数が増えても同様の効果を発揮することができる。ただし，上記のようなカラーセンサを用いる場合は，センサ面に入射する光量の利用効率は１／４に低下する。回折格子を用いる実施例１の回折効率５０％程度の場合と比較すると，実効的な効率は約半分となる。しかしながら，装置構成は非常に単純であり，一層の小型化が可能である。 The overall size of the above fluorescence detection device is based on the volume of a rectangular parallelepiped (18 mm 2 ) defined by the width in the long axis direction of the capillary being 3 mm, the width in the direction perpendicular to the array plane being 2 mm, and the width in the direction of the emission point array being 3 mm. Is also small. That is, the overall size of the fluorescence detection device can be reduced by 1/89,000 times as compared with the case of Patent Document 1. Moreover, since all the optical elements used are minute, it is possible to significantly reduce the cost. Furthermore, the multicolor detection sensitivity of each emission emitted by this fluorescence detection device is high and uniform, the multicolor discrimination accuracy is high, and the crosstalk is low. In the above embodiment, the number of light emitting points was n = 4, but the number is not limited, and the same effect can be exhibited even if the number increases. However, when a color sensor as described above is used, the utilization efficiency of the amount of light incident on the sensor surface is reduced to 1/4. Compared with the case where the diffraction efficiency of Example 1 using the diffraction grating is about 50%, the effective efficiency is about half. However, the device configuration is very simple, and further miniaturization is possible.

センサ面の入射する光量の利用効率を向上するためには，各色を検出する素子が図１２のようにセンサ面と平行に配列するカラーセンサではなく，センサ面に垂直に配列するカラーセンサを用いることが有効である。この場合は必ずしもＳ＜Ｄを満たす必要はない。 In order to improve the utilization efficiency of the amount of light incident on the sensor surface, a color sensor in which the elements for detecting each color are arranged perpendicular to the sensor surface is used instead of the color sensor arranged in parallel with the sensor surface as shown in FIG. Is effective. In this case, it is not always necessary to satisfy S <D.

［実施例４］
本発明の実装上の課題のひとつは，各発光点と各集光レンズの位置合わせを如何に精度良く，簡便に行うかである。本実施例は，複数のキャピラリについて，これを実現する手段を示すものである。 [Example 4]
One of the problems in implementing the present invention is how to accurately and easily align each light emitting point with each condenser lens. This embodiment shows the means to realize this for a plurality of capillaries.

図１３は，複数のキャピラリと，複数のキャピラリを配列するＶ溝アレイと，個別集光レンズアレイとを一体化したデバイスの構成例を示す断面模式図である。図１３（ａ）はレーザ照射位置における各キャピラリの長軸に垂直な断面を示し，図１３（ｂ）はレーザ照射位置ではない個所における各キャピラリの長軸に垂直な断面を示し，図１３（ｃ）は任意の一つのキャピラリの長軸に平行な断面を示す。図１３（ａ）は図１３（ｃ）のＡ−Ａ断面に相当し，図１３（ｂ）は図１３（ｃ）のＢ−Ｂ断面に相当する。 FIG. 13 is a schematic cross-sectional view showing a configuration example of a device in which a plurality of capillaries, a V-groove array in which a plurality of capillaries are arranged, and an individual condenser lens array are integrated. FIG. 13 (a) shows a cross section perpendicular to the long axis of each capillary at the laser irradiation position, and FIG. 13 (b) shows a cross section perpendicular to the long axis of each capillary at a position other than the laser irradiation position. c) shows a cross section parallel to the long axis of any one capillary. 13 (a) corresponds to the AA cross section of FIG. 13 (c), and FIG. 13 (b) corresponds to the BB cross section of FIG. 13 (c).

図１３に示すデバイスは，複数のキャピラリ１からなるキャピラリアレイと，サブデバイス８０を含む。サブデバイス８０は，複数のＶ溝８１が間隔ｐで配列したＶ溝アレイと，複数の集光レンズ１８が間隔ｐで配列した集光レンズアレイが同一デバイスに形成されたものであり，各Ｖ溝８１と各集光レンズ１８の中心軸を一致させてある。複数のキャピラリ１をそれぞれＶ溝８１に押し当てることによって，簡便に，複数のキャピラリ１を所定の間隔ｐで同一平面上に配列させることができる。また，各キャピラリ１のレーザビーム１１の照射位置である各発光点１５と，各集光レンズ１８の焦点が一致するように，サブデバイス８０における各Ｖ溝８１と各集光レンズ１８の相対位置を調整しておく。これにより，発光点１５からの発光が集光レンズ１８によって平行光束１９に変換される。 The device shown in FIG. 13 includes a capillary array composed of a plurality of capillaries 1 and a sub-device 80. The sub-device 80 is a V-groove array in which a plurality of V-grooves 81 are arranged at intervals p, and a condenser lens array in which a plurality of condenser lenses 18 are arranged at intervals p, and each V is formed in the same device. The groove 81 and the central axis of each condenser lens 18 are aligned with each other. By pressing the plurality of capillaries 1 against the V-groove 81, the plurality of capillaries 1 can be easily arranged on the same plane at predetermined intervals p. Further, the relative positions of the V-grooves 81 and the condenser lenses 18 in the subdevice 80 so that the light emitting points 15 which are the irradiation positions of the laser beam 11 of each capillary 1 and the focal points of the condenser lenses 18 coincide with each other. Have been adjusted. As a result, the light emitted from the light emitting point 15 is converted into the parallel luminous flux 19 by the condenser lens 18.

図１３（ａ）に示すように，発光点１５におけるキャピラリ１の断面には，サブデバイス８０の集光レンズ１８が存在し，Ｖ溝８１が存在しない。一方，図１３（ｂ）に示すように，発光点１５の両脇におけるキャピラリ１の断面には，サブデバイス８０の集光レンズ１８が存在せず，Ｖ溝８１が存在する。図１３（ｃ）はキャピラリ１の長軸方向の断面を示し，サブデバイス８０の中央に集光レンズ１８が存在し，その両脇にＶ溝８１が存在している。これは，Ｖ溝８１によるキャピラリ１の高精度な位置合わせを実現しつつ，発光点１５からの発光の検出をＶ溝８１が邪魔をしないようにする工夫である。以上のようなサブデバイス８０を予め作成しておけば，複数のキャピラリ１をそれぞれ各Ｖ溝８１に押し付けるだけで，各発光点１５と各集光レンズ１８の高精度な位置合わせを簡便に行うことが可能となる。本実施例は，実施例１〜３のいずれの構成とも組み合わせることができる。Ｖ溝アレイと集光レンズアレイを一体化したサブデバイス８０は，射出成形やインプリントのような加工法で一体成形することが可能であり，低コストに量産も可能である。もちろん，Ｖ溝８１と集光レンズ１８を別々に作製してから結合させることでサブデバイス８０を完成させても良い。 As shown in FIG. 13A, the condenser lens 18 of the sub-device 80 is present in the cross section of the capillary 1 at the light emitting point 15, and the V-groove 81 is not present. On the other hand, as shown in FIG. 13B, the condenser lens 18 of the sub-device 80 does not exist and the V-groove 81 exists in the cross section of the capillary 1 on both sides of the light emitting point 15. FIG. 13C shows a cross section of the capillary 1 in the long axis direction, in which a condenser lens 18 is present in the center of the subdevice 80, and V grooves 81 are present on both sides thereof. This is a device for preventing the V-groove 81 from interfering with the detection of light emission from the light-emitting point 15 while realizing highly accurate positioning of the capillary 1 by the V-groove 81. If the sub-device 80 as described above is created in advance, high-precision alignment of each light emitting point 15 and each condensing lens 18 can be easily performed by simply pressing a plurality of capillaries 1 against each V-groove 81. It becomes possible. This embodiment can be combined with any of the configurations of Examples 1 to 3. The sub-device 80 that integrates the V-groove array and the condenser lens array can be integrally molded by a processing method such as injection molding or imprinting, and can be mass-produced at low cost. Of course, the sub-device 80 may be completed by separately manufacturing the V-groove 81 and the condenser lens 18 and then coupling them.

サブデバイスはＶ溝アレイが無い場合も有効である。例えば，サブデバイスのキャピラリ配列側の表面をＶ溝アレイではなく，平面としても良い。複数のキャピラリの配列間隔は別の手段によって調整する必要があるが，各キャピラリをサブデバイスの上記平面に押し付けることによって，各キャピラリと各集光レンズの距離，すなわち各発光点と各集光レンズの距離を制御することは可能である。あるいは，Ｖ溝ではなくても，キャピラリの位置を制御するための構造物をサブデバイスに設ければ良い。 The subdevice is also effective in the absence of a V-groove array. For example, the surface of the sub-device on the capillary array side may be a flat surface instead of the V-groove array. The arrangement spacing of multiple capillaries needs to be adjusted by another means, but by pressing each capillary against the above plane of the subdevice, the distance between each capillary and each condenser lens, that is, each emission point and each condenser lens. It is possible to control the distance of. Alternatively, the sub-device may be provided with a structure for controlling the position of the capillary, even if it is not a V-groove.

各集光レンズ１８の，発光点アレイ方向の焦点距離ｆ１と，キャピラリの長軸方向の焦点距離ｆ２とするとき，以上の実施例ではｆ１＝ｆ２としていたが，ｆ１≠ｆ２とすることも有効である。キャピラリ１は円筒形状をしているため，発光点アレイ方向にレンズ作用を持つが，長軸方向にはレンズ作用を持たない。したがって，発光点１５からの発光を集光レンズ１８で効率良く集光するためには，上記のキャピラリのレンズ作用の方向による違いをキャンセルすることが有効であり，そのためにはｆ１≠ｆ２とすれば良い。これは，各集光レンズ１８を非球面とすることで簡単に実現できる。また，各集光レンズ１８をフレネルレンズとすることによって，レンズの厚みを低減し，蛍光検出装置をさらに小型化することも可能である。フレネルレンズの利用は，ｆ１＝ｆ２の場合も，もちろん有効である。 When the focal length f1 in the light emitting point array direction and the focal length f2 in the major axis direction of the capillary of each condenser lens 18 are set, f1 = f2 is set in the above embodiment, but it is also effective to set f1 ≠ f2. Is. Since the capillary 1 has a cylindrical shape, it has a lens action in the light emitting point array direction, but does not have a lens action in the long axis direction. Therefore, in order to efficiently collect the light emitted from the light emitting point 15 by the condenser lens 18, it is effective to cancel the difference depending on the direction of the lens action of the capillary, and for that purpose, f1 ≠ f2. It's fine. This can be easily realized by making each condenser lens 18 an aspherical surface. Further, by using each condenser lens 18 as a Fresnel lens, it is possible to reduce the thickness of the lens and further reduce the size of the fluorescence detection device. Of course, the use of a Fresnel lens is effective even when f1 = f2.

図１４は，複数のキャピラリにそれぞれ個別集光レンズを接着したデバイスの構成例を示す断面模式図である。図１４（ａ）はレーザ照射位置における各キャピラリの長軸に垂直な断面を示し，図１４（ｃ）は任意の一つのキャピラリの長軸に平行な断面を示す。ここでは，各発光点と各集光レンズの位置合わせを精度良く，簡便に行う他の方法を示す。 FIG. 14 is a schematic cross-sectional view showing a configuration example of a device in which individual condenser lenses are bonded to a plurality of capillaries. FIG. 14 (a) shows a cross section perpendicular to the long axis of each capillary at the laser irradiation position, and FIG. 14 (c) shows a cross section parallel to the long axis of any one capillary. Here, we show another method for accurately and easily aligning each light emitting point with each condenser lens.

個別集光レンズ１８を各キャピラリ１に接着し，各集光レンズ１８の焦点と各キャピラリ１の発光点１５が一致するようにする。図１４では，球状の集光レンズ１８を用いているが，もちろん他の形状の集光レンズでも構わない。各集光レンズ１８の各キャピラリ１への接着は，複数のキャピラリの同一平面上への配列が終わってから行う方が望ましい。これは，複数のキャピラリ１の配列工程で，複数の集光レンズ１８が同一平面上に並ばなかったり，複数の集光レンズ１８の光軸が互いに平行でなくなったりすることを避けられる効果を生む。また，上記の接着が終了した状態のキャピラリアレイをユーザに供給することによって，キャピラリアレイの搬送工程，蛍光検出装置への設置工程等で，各発光点１５と各集光レンズ１８の相対位置が所定の位置からずれることを防ぐことができる。 The individual condenser lenses 18 are adhered to each capillary 1 so that the focal point of each condenser lens 18 and the light emitting point 15 of each capillary 1 coincide with each other. In FIG. 14, a spherical condenser lens 18 is used, but of course, a condenser lens having another shape may be used. Adhesion of each condenser lens 18 to each capillary 1 is preferably performed after the arrangement of the plurality of capillaries on the same plane is completed. This has the effect of preventing the plurality of condenser lenses 18 from not lining up on the same plane and the optical axes of the plurality of condenser lenses 18 not being parallel to each other in the process of arranging the plurality of capillaries 1. .. Further, by supplying the user with the capillary array in the state where the above-mentioned adhesion is completed, the relative positions of the light emitting points 15 and the condenser lenses 18 are set in the transfer process of the capillary array, the installation process in the fluorescence detection device, and the like. It is possible to prevent deviation from a predetermined position.

［実施例５］
図１５は，マルチチャンネルを有するマイクロチップと個別集光レンズアレイが一体化したデバイスの構成例を示す断面模式図である。本実施例は，複数のキャピラリではなく，マイクロチップ８６に設けられた複数のチャンネル８２，すなわちチャンネルアレイを対象とする。 [Example 5]
FIG. 15 is a schematic cross-sectional view showing a configuration example of a device in which a microchip having a multi-channel and an individual condenser lens array are integrated. This embodiment targets a plurality of channels 82 provided on the microchip 86, that is, a channel array, instead of a plurality of capillaries.

図示の例のマイクロチップ８６は，表面に複数の角型の４個の溝が形成されたチャンネル基板８３と，表面が平面である平面基板８４を，それぞれの表面を対向させて張り合わせて作製される。チャンネル基板８３と平面基板８４の境界を張り合わせ面８５と呼ぶ。上記の４個の溝は，張り合わせ面８５で仕切られることによって，４個のチャンネル８２を形成する。これらのチャンネル８２は，径０．０５ｍｍであり，間隔ｐ＝０．５ｍｍで同一平面上に配列する。本実施例では，複数のチャンネルの配列平面を，単に配列平面と呼ぶ。径０．０５ｍｍに絞ったレーザビーム１１を配列平面側方より照射することで，数ｎ＝４個，有効径ｄ＝０．０５ｍｍの発光点１５が間隔ｐ＝０．５ｍｍで配列する発光点アレイを得る。発光点アレイの全幅はＷ＝ｐ＊（ｎ−１）＝１．５ｍｍである。本実施例では，さらに，チャンネル基板８３の溝が形成された表面と反対側の裏面に４個の個別集光レンズ１８を形成する。これらの集光レンズ１８は，間隔ｐ＝０．５ｍｍで配列平面と平行に，各光軸が配列平面と垂直になるように配列され，かつ各焦点が各発光点と一致するようにする。 The microchip 86 of the illustrated example is manufactured by laminating a channel substrate 83 having a plurality of square grooves formed on its surface and a flat substrate 84 having a flat surface facing each other. NS. The boundary between the channel substrate 83 and the flat substrate 84 is referred to as a laminated surface 85. The above four grooves are partitioned by the laminating surface 85 to form the four channels 82. These channels 82 have a diameter of 0.05 mm and are arranged on the same plane with a spacing p = 0.5 mm. In this embodiment, the array plane of a plurality of channels is simply referred to as an array plane. By irradiating the laser beam 11 focused to a diameter of 0.05 mm from the side of the array plane, emission points 15 having several n = 4 and an effective diameter d = 0.05 mm are arranged at intervals p = 0.5 mm. Get an array. The total width of the emission point array is W = p * (n-1) = 1.5 mm. In this embodiment, four individual condenser lenses 18 are further formed on the back surface of the channel substrate 83 opposite to the front surface on which the groove is formed. These condenser lenses 18 are arranged so that each optical axis is perpendicular to the array plane and each focal point coincides with each emission point in parallel with the array plane at an interval p = 0.5 mm.

チャンネル基板８３を射出成形やインプリントで作製すれば，表面に溝，裏面に集光レンズ１８を，それぞれの相対位置を上記の通りに精度良く合わせながら低コストに加工することが可能である。ここでは，集光レンズ１８の焦点距離ｆ＝１ｍｍ，有効径Ｄ＝０．４ｍｍとする。集光レンズ１８によって各発光点１５からの発光をそれぞれ集光して平行光束１９とする。それ以降は，これまで説明した，いずれの実施例の蛍光検出装置とも組み合わせることができる。各チャンネル８２を用いて，上記の実施例と同様に電気泳動によるＤＮＡシーケンスを行っても良いし，その他のアプリケーションに適用しても構わない。いずれの場合にも，４個の発光点からの発光を，従来と比較して大幅に小型化された蛍光検出装置を用いて，低クロストークかつ高感度に多色検出できる。 If the channel substrate 83 is manufactured by injection molding or imprinting, it is possible to process a groove on the front surface and a condenser lens 18 on the back surface at low cost while accurately aligning their relative positions as described above. Here, the focal length f = 1 mm and the effective diameter D = 0.4 mm of the condenser lens 18. The light emitted from each light emitting point 15 is collected by the condenser lens 18 to obtain a parallel luminous flux 19. After that, it can be combined with the fluorescence detection device of any of the embodiments described above. Each channel 82 may be used for DNA sequencing by electrophoresis in the same manner as in the above embodiment, or may be applied to other applications. In either case, the emission from the four emission points can be detected in multiple colors with low crosstalk and high sensitivity by using a fluorescence detection device that is significantly smaller than the conventional one.

次に，マイクロチップ８６を用いた，より具体的な実施例を説明する。 Next, a more specific embodiment using the microchip 86 will be described.

図１６は，マルチチャンネルを有するマイクロチップと個別集光レンズアレイが一体化したデバイスと個別ＬＥＤ照明による発光点アレイからの発光を，共通ダイクロイックミラーセット，及びセンサにより多色検出する装置構成例を示す断面模式図である。図１６（ａ）はマイクロチップの上面模式図，図１６（ｂ）は任意の１個のチャンネルを側面から見た断面模式図，図１６（ｃ）は，蛍光標識されてチャンネル内部を流れる液滴と発光点の関係を示す説明図である。 FIG. 16 shows an example of a device configuration in which a device in which a microchip having a multi-channel and an individual condensing lens array are integrated and light emission from a light emitting point array by individual LED lighting are detected in multiple colors by a common dichroic mirror set and a sensor. It is a cross-sectional schematic diagram which shows. 16 (a) is a schematic top view of a microchip, FIG. 16 (b) is a schematic cross-sectional view of any one channel viewed from the side, and FIG. 16 (c) is a fluorescently labeled liquid flowing inside the channel. It is explanatory drawing which shows the relationship between a drop and a light emitting point.

図１６（ａ）の上面模式図に示すように，図示の例のマイクロチップ８６には，径０．１ｍｍ，長さ５０ｍｍの１０個のチャンネル８２が，間隔ｐ＝２ｍｍで，平行かつ同一平面上に配列している。各チャンネル８２の両端には，フロー入口８７とフロー出口８８が形成されている。各チャンネル８２の中央に発光点１５が位置している。 As shown in the schematic top view of FIG. 16 (a), in the microchip 86 of the illustrated example, ten channels 82 having a diameter of 0.1 mm and a length of 50 mm are arranged in parallel and in the same plane with a spacing of p = 2 mm. Arranged above. A flow inlet 87 and a flow outlet 88 are formed at both ends of each channel 82. The light emitting point 15 is located in the center of each channel 82.

図１６（ｂ）に示すように，これまでの実施例と異なり，本実施例では，励起光としてレーザビームの代わりにＬＥＤ光を用い，落射蛍光検出の光学系を採用する。また，ＬＥＤ光源９０は，チャンネル８２毎に個別に準備する。個別ＬＥＤ光源９０から発振した中心波長５０５ｎｍのＬＥＤ光は，焦点距離５ｍｍの個別コリメートレンズ９１によりＬＥＤ平行光束９２とされた後，共通ＬＥＤダイクロイックミラー８９に４５°の入射角で並列に入射して反射され，配列平面に向かって垂直に進む。ＬＥＤダイクロイックミラー８９の中心は，各集光レンズ１８から１ｍｍの距離の位置に配置する。次に，各ＬＥＤ平行光束９２は，焦点距離ｆ＝１ｍｍ，有効径Ｄ＝１ｍｍの個別集光レンズ１８によって各発光点１５の位置にそれぞれ集光される。このとき，ＬＥＤ光の集光サイズは径０．０５ｍｍとなるため，発光点のサイズもｄ＝０．０５ｍｍとなり，チャンネル８２の径０．１ｍｍよりも小さくすることができる。これはクロストークを低減する上で有利となる。 As shown in FIG. 16B, unlike the previous embodiments, in this embodiment, LED light is used instead of the laser beam as the excitation light, and an optical system for detecting epi-fluorescence is adopted. Further, the LED light source 90 is individually prepared for each channel 82. The LED light with a center wavelength of 505 nm oscillated from the individual LED light source 90 is made into an LED parallel light beam 92 by the individual collimated lens 91 having a focal length of 5 mm, and then incidents in parallel on the common LED dichroic mirror 89 at an incident angle of 45 °. It is reflected and travels vertically toward the array plane. The center of the LED dichroic mirror 89 is arranged at a distance of 1 mm from each condenser lens 18. Next, each LED parallel light beam 92 is focused at the position of each light emitting point 15 by an individual condenser lens 18 having a focal length f = 1 mm and an effective diameter D = 1 mm. At this time, since the condensing size of the LED light is 0.05 mm in diameter, the size of the light emitting point is also d = 0.05 mm, which can be smaller than the diameter of 0.1 mm of the channel 82. This is advantageous in reducing crosstalk.

各発光点１５からの発光は，同一の個別集光レンズ１８によりそれぞれ平行光束１９とされ，共通ＬＥＤダイクロイックミラー８９に４５°の入射角で並列に入射し，ＬＥＤ光はＬＥＤダイクロイックミラー８９でそれぞれ反射してＬＥＤ光源９０の方向に進み，蛍光はＬＥＤダイクロイックミラー８９をそれぞれ透過する。以降の，Ａダイクロイックミラー３９，Ｂダイクロイックミラー４１，Ｃダイクロイックミラー４３，及びＤダイクロイックミラー４５の各要素を各発光点について共通かつ並列に用い，Ａ蛍光，Ｂ蛍光，Ｃ蛍光，及びＤ蛍光を検出する点は実施例２と同等である。実施例２と異なる点は，Ａダイクロイックミラー３９の中心と各集光レンズ１８の距離が２ｍｍであること，各ダイクロイックミラー３９，４１，４３，４５の発光点アレイの配列方向の有効径がＤＭ１＝２５ｍｍであることである。このとき，各集光レンズ１８とセンサ面２７の距離はｇ＝６ｍｍである。ｆ＝１ｍｍに対して，−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ＝２．７４ｍｍとなり，式（１）が満足され，相対検出光量は３６２％（＞５０％）となる。また，０．９５＊（ｄ／ｐ）＊ｇ＝０．１４ｍｍとなり，式（２）が満足され，クロストーク信号強度比が０．０％（＜２５％）となる。 The light emitted from each light emitting point 15 is made into a parallel light beam 19 by the same individual condensing lens 18, and is incident in parallel on the common LED dichroic mirror 89 at an incident angle of 45 °, and the LED light is incident on the LED dichroic mirror 89, respectively. It reflects and travels in the direction of the LED light source 90, and the fluorescence passes through the LED dichroic mirror 89, respectively. Subsequent elements of the A dichroic mirror 39, the B dichroic mirror 41, the C dichroic mirror 43, and the D dichroic mirror 45 are used in common and in parallel for each emission point, and A fluorescence, B fluorescence, C fluorescence, and D fluorescence are used. The points to be detected are the same as those in the second embodiment. The difference from the second embodiment is that the distance between the center of the A dichroic mirror 39 and each condenser lens 18 is 2 mm, and the effective diameter of the emission point arrays of the dichroic mirrors 39, 41, 43, 45 in the arrangement direction is DM1. = 25 mm. At this time, the distance between each condenser lens 18 and the sensor surface 27 is g = 6 mm. For f = 1 mm, −0.20 * (d / D) * g + 2.8 * D = 2.74 mm, the equation (1) is satisfied, and the relative detection light amount is 362% (> 50%). .. Further, 0.95 * (d / p) * g = 0.14 mm, the equation (2) is satisfied, and the crosstalk signal intensity ratio is 0.0% (<25%).

本実施例では，上記のマイクロチップ８６及び蛍光検出装置をデジタルＰＣＲの測定に用いる。デジタルＰＣＲでは，オイル中に多数の液滴（エマルジョン）を形成し，各液滴にターゲットのＤＮＡ分子が０個か１個だけ含まれるようにする。この状態でＰＣＲを行い，ターゲットが存在して増幅された場合に蛍光を発光するようにする。ひとつひとつの液滴の蛍光発光の有無を調べることによって，元のサンプル中に存在したターゲットの分子数を高精度に定量する。さらに，４色の蛍光検出を行うことによって，４種類のターゲットについて，独立にデジタルＰＣＲを行うことが可能である。デジタルＰＣＲの課題の一つはスループットの向上であり，多数の液滴の高スループットな多色検出が重要である。図１６（ｃ）は，チャンネル８２の内部を，オイルとともに，Ａ蛍光，Ｂ蛍光，Ｃ蛍光，及びＤ蛍光で標識された４種類の液滴９３，９４，９５，９６がフローし，発光点１５を横切る際に励起を受けて各蛍光を発光する構成を示している。従来は，１個のチャンネルを用いた１色検出によりデジタルＰＣＲの測定を行っていたが，本実施例では，１０個のチャンネルを用いた４色検出によりデジタルＰＣＲの測定を行えるため，スループットは４０倍となる。しかも，マイクロチップ８６及び蛍光検出装置は非常に小型であり，低コストに製造可能である。 In this example, the above-mentioned microchip 86 and fluorescence detection device are used for digital PCR measurement. In digital PCR, a large number of droplets (emulsions) are formed in the oil so that each droplet contains only 0 or 1 target DNA molecule. PCR is performed in this state so that fluorescence is emitted when the target is present and amplified. By examining the presence or absence of fluorescence emission of each droplet, the number of molecules of the target present in the original sample is quantified with high accuracy. Furthermore, by performing fluorescence detection of four colors, it is possible to independently perform digital PCR on four types of targets. One of the challenges of digital PCR is to improve throughput, and high-throughput multicolor detection of a large number of droplets is important. In FIG. 16 (c), four types of droplets 93, 94, 95, 96 labeled with A fluorescence, B fluorescence, C fluorescence, and D fluorescence flow inside the channel 82 together with oil, and the emission point. A configuration is shown in which each fluorescence is emitted by being excited when crossing 15. In the past, digital PCR was measured by one-color detection using one channel, but in this example, digital PCR can be measured by four-color detection using ten channels, so the throughput is high. It will be 40 times. Moreover, the microchip 86 and the fluorescence detector are very small and can be manufactured at low cost.

なお、本発明は上記した実施例に限定されるものではなく、様々な変形例が含まれる。例えば、上記した実施例は本発明を分かりやすく説明するために詳細に説明したものであり、必ずしも説明した全ての構成を備えるものに限定されるものではない。また、ある実施例の構成の一部を他の実施例の構成に置き換えることが可能であり、また、ある実施例の構成に他の実施例の構成を加えることも可能である。また、各実施例の構成の一部について、他の構成の追加・削除・置換をすることが可能である。 The present invention is not limited to the above-described embodiment, and includes various modifications. For example, the above-described embodiment has been described in detail in order to explain the present invention in an easy-to-understand manner, and is not necessarily limited to the one including all the described configurations. Further, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Further, it is possible to add / delete / replace a part of the configuration of each embodiment with another configuration.

１ キャピラリ
１０ レーザ光源
１１ レーザビーム
１２ レーザ照射位置
１５ 発光点
１７ 集光レンズアレイ
１８ 集光レンズ
２０ ロングパスフィルタ
２１ 透過型回折格子
２３ 結像レンズ
２７ センサ面
２８ センサ領域
３８ 凹面反射型回折格子
３９ Ａダイクロイックミラー
４１ Ｂダイクロイックミラー
４３ Ｃダイクロイックミラー
４５ Ｄダイクロイックミラー
４７ 波長分散像
５６ ロングパスフィルタ
７３ カラーセンサ面
８０ サブデバイス
８１ Ｖ溝
８２ チャンネル
８３ チャンネル基板
８４ 平面基板
８６ マイクロチップ
９０ ＬＥＤ光源
９１ コリメートレンズ
９７ 低分散プリズム 1 Capillary 10 Laser light source 11 Laser beam 12 Laser irradiation position 15 Light emitting point 17 Condensing lens array 18 Condensing lens 20 Long pass filter 21 Transmission type diffraction grid 23 Imaging lens 27 Sensor surface 28 Sensor area 38 Concave reflection type diffraction grid 39 A Dichroic Mirror 41 B Dichroic Mirror 43 C Dichroic Mirror 45 D Dichroic Mirror 47 Wavelength Dispersion Image 56 Long Path Filter 73 Color Sensor Surface 80 Subdevice 81 V Groove 82 Channel 83 Channel Board 84 Flat Board 86 Microchip 90 LED Light Source 91 Collimated Lens 97 Low Dispersion prism

Claims (22)

ｍ，ｎをそれぞれ２以上の任意の整数として，
ｍ個の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に集光してｍ個の光束とするｍ個の集光レンズが配列した集光レンズアレイと，
少なくとも第１のダイクロイックミラーと第２のダイクロイックミラーを含む，ｎ個のダイクロイックミラーが略平行に配列したダイクロイックミラーアレイと，
少なくとも１個のセンサと，を備え，
前記ｍ個の光束の少なくとも一部が前記第１のダイクロイックミラーにそれぞれ並列に入射され，前記第１のダイクロイックミラーが前記ｍ個の光束の少なくとも一部をそれぞれ，ｍ個の第１の透過光束と，ｍ個の第１の反射光束に分割し，
前記ｍ個の第１の反射光束の少なくとも一部が前記第２のダイクロイックミラーにそれぞれ並列に入射され，前記第２のダイクロイックミラーが前記ｍ個の第１の反射光束の少なくとも一部をそれぞれ，少なくともｍ個の第２の反射光束に変換し，
前記ｍ個の第１の透過光束の少なくとも一部と前記ｍ個の第２の反射光束の少なくとも一部が，再集光されずに，前記センサにそれぞれ並列に入射される多色検出装置。 Let m and n be arbitrary integers of 2 or more, respectively.
A condenser lens array in which m condenser lenses are arranged to individually collect light emitted from each emission point of the emission point array in which m emission points are arranged to obtain m luminous flux.
A dichroic mirror array in which n dichroic mirrors are arranged substantially in parallel, including at least a first dichroic mirror and a second dichroic mirror.
With at least one sensor,
At least a part of the m light flux is incident on the first dichroic mirror in parallel, and the first dichroic mirror makes at least a part of the m light flux, each of which is m first transmitted light flux. And divided into m first reflected luminous flux,
At least a part of the m first reflected light flux is incident on the second dichroic mirror in parallel, and the second dichroic mirror makes at least a part of the m first reflected light flux, respectively. Converted to at least m second reflected light flux,
A multicolor detection device in which at least a part of the m first transmitted light flux and at least a part of the m second reflected light flux are incident on the sensor in parallel without being refocused. 請求項１に記載の多色検出装置において，
前記ｍ個の集光レンズの光軸の方向と，前記センサのセンサ面が略垂直である多色検出装置。 In the multicolor detection device according to claim 1,
A multicolor detection device in which the direction of the optical axis of the m condenser lenses and the sensor surface of the sensor are substantially perpendicular to each other. 請求項１又は２に記載の多色検出装置において，
前記ｍ個の集光レンズの光軸の方向と，前記ｍ個の集光レンズが配列する方向のそれぞれと，前記ｎ個のダイクロイックミラーが配列する方向が略垂直である多色検出装置。 In the multicolor detection device according to claim 1 or 2.
A multicolor detection device in which the direction of the optical axis of the m condensing lenses, the direction in which the m condensing lenses are arranged, and the direction in which the n dichroic mirrors are arranged are substantially perpendicular to each other. 請求項１に記載の多色検出装置において，
前記ｍ個の発光点の有効径の平均をｄ，前記ｍ個の集光レンズの焦点距離の平均をｆ，前記ｍ個の集光レンズの有効径の平均をＤ，前記ｍ個の第２の反射光束の，前記ｍ個の集光レンズと前記センサの間の光路長の平均をｇとするとき，
ｆ≦−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ
を満足する多色検出装置。 In the multicolor detection device according to claim 1,
The average of the effective diameters of the m light emitting points is d, the average of the focal lengths of the m condenser lenses is f, the average of the effective diameters of the m condenser lenses is D, and the average of the m second condenser lenses is d. When the average of the optical path lengths between the m condensing lenses and the sensor of the reflected light beam is g.
f ≦ -0.20 * (d / D) * g + 2.8 * D
A multicolor detector that satisfies. 請求項４に記載の多色検出装置において，
前記ｍ個の発光点の配列間隔の平均をｐとするとき，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ
を満足する多色検出装置。 In the multicolor detection device according to claim 4,
When the average of the arrangement intervals of the m light emitting points is p,
f ≧ 0.95 * (d / p) * g
A multicolor detector that satisfies. 請求項１に記載の多色検出装置において，
前記ｍ個の発光点の有効径の平均をｄ，前記ｍ個の集光レンズの焦点距離の平均をｆ，前記ｍ個の発光点の配列間隔の平均をｐ，前記ｍ個の第２の反射光束の，前記ｍ個の集光レンズと前記センサの間の光路長の平均をｇとするとき，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ
を満足する多色検出装置。 In the multicolor detection device according to claim 1,
The average of the effective diameters of the m light emitting points is d, the average of the focal lengths of the m condensing lenses is f, the average of the arrangement intervals of the m light emitting points is p, and the average of the m second light emitting points is p. When the average optical path length between the m condensing lenses and the sensor of the reflected light beam is g.
f ≧ 0.95 * (d / p) * g
A multicolor detector that satisfies. 請求項１に記載の多色検出装置において，
前記ｍ個の発光点の配列間隔の平均をｐ，前記ｍ個の集光レンズの有効径の平均をＤ，前記ｎ個のダイクロイックミラーの前記ｍ個の発光点の配列方向の有効径をＤＭ１，前記ｎ個のダイクロイックミラーの前記ｍ個の発光点の配列方向と直交方向の有効径をＤＭ２とするとき，
ｐ＊（ｍ−１）＋Ｄ≦ＤＭ１，及び
√２＊Ｄ≦ＤＭ２
を満足する多色検出装置。 In the multicolor detection device according to claim 1,
The average of the arrangement intervals of the m light emitting points is p, the average of the effective diameters of the m condensing lenses is D, and the effective diameter of the n dichroic mirrors in the arrangement direction of the m light emitting points is DM1. When the effective diameter of the n dichroic mirrors in the direction orthogonal to the arrangement direction of the m light emitting points is DM2,
p * (m-1) + D≤DM1, and √2 * D≤DM2
A multicolor detector that satisfies. 請求項１に記載の多色検出装置において，
前記ｍ個の集光レンズの光軸が互いに平行ではない多色検出装置。 In the multicolor detection device according to claim 1,
A multicolor detection device in which the optical axes of the m condenser lenses are not parallel to each other. 請求項１に記載の多色検出装置において，
前記ダイクロイックミラーアレイに属さない，第３のダイクロイックミラーをさらに有し，
ｍ個の照射光束が前記第３のダイクロイックミラーにそれぞれ並列に入射され，前記第３のダイクロイックミラーが前記ｍ個の照射光束の少なくとも一部をそれぞれ，少なくともｍ個の第３の反射光束に変換し，
前記ｍ個の第３の反射光束が，前記ｍ個の集光レンズによってそれぞれ個別に集光され，前記ｍ個の発光点をそれぞれ個別に照射し，
前記ｍ個の光束が，前記ｍ個の発光点からの発光を前記ｍ個の集光レンズによってそれぞれ個別に集光し，前記第３のダイクロイックミラーを並列に透過させた光束である多色検出装置。 In the multicolor detection device according to claim 1,
It also has a third dichroic mirror that does not belong to the dichroic mirror array.
The m irradiation light fluxes are incident on the third dichroic mirror in parallel, and the third dichroic mirror converts at least a part of the m irradiation light fluxes into at least m third reflected light fluxes, respectively. death,
The m third reflected light fluxes are individually focused by the m condensing lenses, and the m emission points are individually irradiated.
Multicolor detection, which is a luminous flux in which the m light beams individually focus the light emitted from the m light emitting points by the m condenser lenses and transmit the third dichroic mirror in parallel. Device. ｍ，ｎをそれぞれ２以上の任意の整数として，
ｍ個の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に集光してｍ個の光束とするｍ個の集光レンズが配列した集光レンズアレイと，
少なくとも第１のダイクロイックミラーと第２のダイクロイックミラーを含む，ｎ個のダイクロイックミラーが略平行に配列したダイクロイックミラーアレイと，
少なくとも１個のセンサと，を備え，
前記ｍ個の光束の少なくとも一部が前記第１のダイクロイックミラーにそれぞれ並列に入射され，前記第１のダイクロイックミラーが前記ｍ個の光束の少なくとも一部をそれぞれ，ｍ個の第１の透過光束と，ｍ個の第１の反射光束に分割し，
前記ｍ個の第１の透過光束の少なくとも一部が前記第２のダイクロイックミラーにそれぞれ並列に入射され，前記第２のダイクロイックミラーが前記ｍ個の第１の透過光束の少なくとも一部をそれぞれ，少なくともｍ個の第２の反射光束に変換し，
前記ｍ個の第１の反射光束の少なくとも一部と前記ｍ個の第２の反射光束の少なくとも一部が，再集光されずに，前記センサにそれぞれ並列に入射される多色検出装置。 Let m and n be arbitrary integers of 2 or more, respectively.
A condenser lens array in which m condenser lenses are arranged to individually collect light emitted from each emission point of the emission point array in which m emission points are arranged to obtain m luminous flux.
A dichroic mirror array in which n dichroic mirrors are arranged substantially in parallel, including at least a first dichroic mirror and a second dichroic mirror.
With at least one sensor,
At least a part of the m light flux is incident on the first dichroic mirror in parallel, and the first dichroic mirror makes at least a part of the m light flux, each of which is m first transmitted light flux. And divided into m first reflected luminous flux,
At least a part of the m first transmitted light flux is incident on the second dichroic mirror in parallel, and the second dichroic mirror makes at least a part of the m first transmitted light flux, respectively. Converted to at least m second reflected light flux,
A multicolor detection device in which at least a part of the m first reflected light flux and at least a part of the m second reflected light flux are incident on the sensor in parallel without being refocused. 請求項１０に記載の多色検出装置において，
前記ｍ個の集光レンズの光軸の方向と，前記センサのセンサ面が略垂直である多色検出装置。 In the multicolor detection device according to claim 10,
A multicolor detection device in which the direction of the optical axis of the m condenser lenses and the sensor surface of the sensor are substantially perpendicular to each other. 請求項１０又は１１に記載の多色検出装置において，
前記ｍ個の集光レンズの光軸の方向と，前記ｍ個の集光レンズが配列する方向のそれぞれと，前記ｎ個のダイクロイックミラーが配列する方向が略垂直である多色検出装置。 In the multicolor detection device according to claim 10 or 11.
A multicolor detection device in which the direction of the optical axis of the m condensing lenses, the direction in which the m condensing lenses are arranged, and the direction in which the n dichroic mirrors are arranged are substantially perpendicular to each other. 請求項１０に記載の多色検出装置において，
前記ｍ個の発光点の有効径の平均をｄ，前記ｍ個の集光レンズの焦点距離の平均をｆ，前記ｍ個の集光レンズの有効径の平均をＤ，前記ｍ個の第２の反射光束の，前記ｍ個の集光レンズと前記センサの間の光路長の平均をｇとするとき，
ｆ≦−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ
を満足する多色検出装置。 In the multicolor detection device according to claim 10,
The average of the effective diameters of the m light emitting points is d, the average of the focal lengths of the m condenser lenses is f, the average of the effective diameters of the m condenser lenses is D, and the average of the m second condenser lenses is d. When the average of the optical path lengths between the m condensing lenses and the sensor of the reflected light beam is g.
f ≦ -0.20 * (d / D) * g + 2.8 * D
A multicolor detector that satisfies. 請求項１３に記載の多色検出装置において，
前記ｍ個の発光点の配列間隔の平均をｐとするとき，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ
を満足する多色検出装置。 In the multicolor detection device according to claim 13,
When the average of the arrangement intervals of the m light emitting points is p,
f ≧ 0.95 * (d / p) * g
A multicolor detector that satisfies. 請求項１０に記載の多色検出装置において，
前記ｍ個の発光点の有効径の平均をｄ，前記ｍ個の発光点の配列間隔の平均をｐ，前記ｍ個の集光レンズの焦点距離の平均をｆ，前記ｍ個の第２の反射光束の，前記ｍ個の集光レンズと前記センサの間の光路長の平均をｇとするとき，
ｆ≧０．９５＊（ｄ／ｐ）＊ｇ
を満足する多色検出装置。 In the multicolor detection device according to claim 10,
The average of the effective diameters of the m light emitting points is d, the average of the arrangement intervals of the m light emitting points is p, the average of the focal lengths of the m light emitting lenses is f, and the average of the m second light emitting points is the second. When the average optical path length between the m condensing lenses and the sensor of the reflected light beam is g.
f ≧ 0.95 * (d / p) * g
A multicolor detector that satisfies. 請求項１０に記載の多色検出装置において，
前記ｍ個の発光点の配列間隔の平均をｐ，前記ｍ個の集光レンズの有効径の平均をＤ，前記ｎ個のダイクロイックミラーの前記ｍ個の発光点の配列方向の有効径をＤＭ１，前記ｎ個のダイクロイックミラーの前記ｍ個の発光点の配列方向と直交方向の有効径をＤＭ２とするとき，
ｐ＊（ｍ−１）＋Ｄ≦ＤＭ１，及び
√２＊Ｄ≦ＤＭ２
を満足する多色検出装置。 In the multicolor detection device according to claim 10,
The average of the arrangement intervals of the m light emitting points is p, the average of the effective diameters of the m condensing lenses is D, and the effective diameter of the n dichroic mirrors in the arrangement direction of the m light emitting points is DM1. When the effective diameter of the n dichroic mirrors in the direction orthogonal to the arrangement direction of the m light emitting points is DM2,
p * (m-1) + D≤DM1, and √2 * D≤DM2
A multicolor detector that satisfies. 請求項１０に記載の多色検出装置において，
前記ｍ個の集光レンズの光軸が互いに平行ではない多色検出装置。 In the multicolor detection device according to claim 10,
A multicolor detection device in which the optical axes of the m condenser lenses are not parallel to each other. 請求項１０に記載の多色検出装置において，
前記ダイクロイックミラーアレイに属さない，第３のダイクロイックミラーをさらに有し，
ｍ個の照射光束が前記第３のダイクロイックミラーにそれぞれ並列に入射され，前記第３のダイクロイックミラーが前記ｍ個の照射光束の少なくとも一部をそれぞれ，少なくともｍ個の第３の反射光束に変換し，
前記ｍ個の第３の反射光束が，前記ｍ個の集光レンズによってそれぞれ個別に集光され，前記ｍ個の発光点をそれぞれ個別に照射し，
前記ｍ個の光束が，前記ｍ個の発光点からの発光を前記ｍ個の集光レンズによってそれぞれ個別に集光し，前記第３のダイクロイックミラーを並列に透過させた光束である多色検出装置。 In the multicolor detection device according to claim 10,
It also has a third dichroic mirror that does not belong to the dichroic mirror array.
The m irradiation light fluxes are incident on the third dichroic mirror in parallel, and the third dichroic mirror converts at least a part of the m irradiation light fluxes into at least m third reflected light fluxes, respectively. death,
The m third reflected light fluxes are individually focused by the m condensing lenses, and the m emission points are individually irradiated.
Multicolor detection, which is a luminous flux in which the m light beams individually focus the light emitted from the m light emitting points by the m condenser lenses and transmit the third dichroic mirror in parallel. Device. ｍを２以上の任意の整数として，
ｍ個の発光点が配列した発光点アレイの各発光点からの発光をそれぞれ個別に集光してｍ個の光束とするｍ個の集光レンズが配列した集光レンズアレイと，
少なくとも１個のセンサと，を備え，
前記ｍ個の光束の少なくとも一部が前記センサにそれぞれ並列に入射され，
前記ｍ個の発光点がそれぞれ有限サイズであり，
前記ｍ個の発光点の有効径の平均をｄ，前記ｍ個の集光レンズの焦点距離の平均をｆ，前記ｍ個の集光レンズの有効径の平均をＤ，前記ｍ個の光束の，前記ｍ個の集光レンズと前記センサの間の光路長の平均をｇとするとき，
ｆ≦−０．２０＊（ｄ／Ｄ）＊ｇ＋２．８＊Ｄ
を満足する多色検出装置。 Let m be any integer greater than or equal to 2
A condenser lens array in which m condenser lenses are arranged to individually collect light emitted from each emission point of the emission point array in which m emission points are arranged to obtain m luminous flux.
With at least one sensor,
At least a part of the m luminous flux is incident on the sensor in parallel, respectively.
Each of the m light emitting points has a finite size.
The average of the effective diameters of the m light emitting points is d, the average of the focal lengths of the m condenser lenses is f, the average of the effective diameters of the m condenser lenses is D, and the average of the m light beams. When the average optical path length between the m condenser lenses and the sensor is g,
f ≦ -0.20 * (d / D) * g + 2.8 * D
A multicolor detector that satisfies. ｍを２以上の任意の整数として，
ｍ個のキャピラリの少なくとも一部が同一平面上に配列したキャピラリアレイと，
前記同一平面上の前記ｍ個のキャピラリからの発光を，それぞれ個別に集光してｍ個の光束とするｍ個の集光レンズが配列した集光レンズアレイと，
１個のセンサと，を備え，
前記ｍ個の光束の少なくとも一部が前記センサに，再集光されずに，それぞれ並列に入射され，
前記同一平面と前記センサのセンサ面が互いに略平行である多色検出装置。 Let m be any integer greater than or equal to 2
A capillary array in which at least a part of m capillaries are arranged on the same plane,
A condensing lens array in which m condensing lenses are arranged to individually condense light emitted from the m capillaries on the same plane into m luminous fluxes.
Equipped with one sensor,
At least a part of the m luminous fluxes are incident on the sensor in parallel without being refocused.
A multicolor detection device in which the same plane and the sensor surface of the sensor are substantially parallel to each other. 請求項２０に記載の多色検出装置において，
前記センサが，異なる分光感度特性を有する複数種類の画素が２次元状に配列して構成されている多色検出装置。 In the multicolor detection device according to claim 20,
The sensor is a multicolor detection device in which a plurality of types of pixels having different spectral sensitivity characteristics are arranged two-dimensionally. 請求項２１に記載の多色検出装置において，
前記ｍ個の集光レンズの有効径の平均をＤ，前記センサの画素サイズの平均をＳとするとき，
Ｓ＜Ｄ
を満足する多色検出装置。 In the multicolor detection device according to claim 21,
When the average of the effective diameters of the m condenser lenses is D and the average of the pixel sizes of the sensors is S,
S <D
A multicolor detector that satisfies.
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