WO2014020967A1 - 共焦点顕微鏡又は多光子顕微鏡の光学系を用いた光分析装置、光分析方法及び光分析用コンピュータプログラム - Google Patents
共焦点顕微鏡又は多光子顕微鏡の光学系を用いた光分析装置、光分析方法及び光分析用コンピュータプログラム Download PDFInfo
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- G01J1/00—Photometry, e.g. photographic exposure meter
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- G01J1/16—Photometry, e.g. photographic exposure meter by comparison with reference light or electric value provisionally void using electric radiation detectors
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Definitions
- the present invention uses an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, and uses atoms, molecules or aggregates thereof dispersed or dissolved in a solution ( These are hereinafter referred to as “particles”), for example, biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, particulate objects such as viruses and cells, or non-
- the present invention relates to an optical analysis technique capable of detecting light from biological particles and obtaining useful information in analysis or analysis of those states (interaction, binding / dissociation state, etc.).
- the detected light may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, or the like.
- the particles that emit light (hereinafter referred to as “luminescent particles”) may be either particles that emit light themselves, or particles to which an arbitrary luminescent label or luminescent probe is added.
- optical analysis techniques include, for example, fluorescence correlation spectroscopy (FCS; see, for example, Patent Document 1-3), fluorescence intensity distribution analysis (Fluorescence-Intensity Distribution Analysis: FIDA, for example, Patent Document 4).
- Patent Documents 6 to 8 propose a method for detecting a fluorescent substance based on the passage of time of a fluorescence signal of a sample solution measured using an optical system of a confocal microscope.
- Patent Documents 9 to 11 is an optical analysis technique using an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope.
- a new optical analysis technique based on a principle different from optical analysis techniques such as FCS and FIDA has been proposed.
- a novel photoanalysis technique hereinafter referred to as “scanning molecule counting method”
- a micro area hereinafter referred to as “light detection area” that is a light detection area in the sample solution is used.
- the light detection region is dispersed in the sample solution while moving the position of the sample solution by the light detection region.
- the light emitted from the luminescent particles is individually detected, thereby detecting the luminescent particles in the sample solution one by one and counting the luminescent particles or in the sample solution. It is possible to obtain information on the concentration or number density of the luminescent particles.
- a sample solution To the photodetector must be in a planned state.
- the sample solution surely exists in the sample container, and the focal region of the objective lens of the optical system, that is, the minute region (light detection region) must surely exist in the sample solution.
- a predetermined liquid water, oil, etc.
- the excitation light must be focused within the sample solution at the planned intensity and at the planned area to form a micro-area.
- a photodetector that detects light from a minute region needs to be in a state that can convert normally received light into an electrical signal.
- the light intensity is measured while moving the position of the light detection area of the objective lens in the sample solution. If there is a part that deviates from the surface or a foreign substance that affects the measurement of light intensity, it may require labor for analysis processing such as processing to exclude the light intensity measurement value of that part. It is preferable that a part deviating from the inside, a foreign object, and the like are not present in advance.
- the measurement of the light intensity may take several tens of minutes to about one hour.) In addition, it may be expensive. Therefore, it is advantageous to be able to determine whether or not a predetermined state is realized in the optical system before measurement without depending on the experience of the user.
- one object of the present invention is an optical analysis technique using the optical system of a confocal microscope or a multiphoton microscope for optical analysis techniques such as the above-mentioned scanning molecule counting method, FCS, FIDA, and PCH. It is possible to determine whether the planned state in the optical system has been realized before measuring the light intensity, without relying on the user's experience or observing the resulting state after measuring the light intensity. It is to provide a new configuration.
- the signal intensity output by the photodetector is generally changed depending on the state of the optical system from the sample solution to the photodetector.
- the size of the background (background intensity) changes. That is, whether or not a state planned in the optical system, that is, a state in which the light detection region is arranged in the sample solution and measurement of the light intensity from the light detection region can be performed is achieved. This can be determined by referring to the magnitude of the signal intensity output by the detector. Such knowledge is used in the present invention.
- the above-described problem is obtained by measuring light intensity from a light detection region arranged in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, and analyzing the light intensity.
- An analysis device a light detection region position mover that moves the position of the light detection region with respect to the sample container, a light detector for detecting light from the light detection region, and a position of the light detection region with respect to the sample container Based on the magnitude of the signal intensity output by the photodetector while being moved, whether the light detection area is arranged in the sample solution and measurement of the light intensity from the light detection area is feasible and the light intensity
- a determination unit for determining at least one of the position of the light detection region relative to the sample container or the range thereof.
- the “light detection region” of the optical system of the confocal microscope or multiphoton microscope is a confocal volume, that is, a minute region in which light is detected in the microscope, and is excited from the objective lens. When light is given, it corresponds to the region where the excitation light is collected.
- the measurement and analysis of the light intensity executed by this apparatus is a light that measures and analyzes the light intensity using an optical system of a confocal microscope or a multiphoton microscope such as a scanning molecule counting method, FCS, FIDA, and PCH. Any analysis technique may be used.
- the position of the light detection region with respect to the sample container is achieved by moving the focal position by moving the sample container or the stage carrying the sample container, or by changing the direction of the optical element in the optical path of the optical system. May be.
- the apparatus of the present invention basically has the light intensity from the light detection region arranged in the sample solution using the optical system of the confocal microscope or the multiphoton microscope described in the above-mentioned patent documents. It is applied to an optical analyzer that performs measurement and analysis. As already mentioned, in the optical system, whether or not the state in which the light detection region is arranged in the sample solution and the measurement of the light intensity from the light detection region is feasible is achieved by the photodetector. This can be determined by referring to the magnitude of the output signal strength.
- the present invention in the microscope, while moving the position of the light detection region with respect to the sample container, referring to the magnitude of the signal intensity output from the photodetector, based on the magnitude of the signal intensity, Whether or not the state in which the light detection region is arranged in the sample solution and the measurement of the light intensity from the light detection region is feasible is achieved, or the state of the light detection region with respect to the sample container in which such a state is achieved The position or its range is determined. According to such a configuration, the magnitude of the signal intensity output from the photodetector can be referred to without depending on the experience of the user or the observation of the result after measurement of the light intensity.
- the light intensity measurement can be performed advantageously.
- the determination device that performs the above determination may typically be realized by an operation according to a computer program of the optical analyzer.
- the determination device performs light detection obtained when the light detection region is arranged in the sample solution and the measurement of the light intensity from the light detection region can be performed in order to perform the above determination.
- the range of the magnitude of the signal strength output from the device (first signal strength reference value range) may be stored. Then, at the time of determination performed while moving the position of the light detection region relative to the sample container, the magnitude of the signal intensity output by the photodetector is referred to, and the magnitude of the signal intensity is the first light intensity reference value. When it is within the range, it may be determined that the measurement of the light intensity from the light detection region is feasible at the position of the light detection region with respect to the sample container at that time.
- the light detection region is arranged in the sample solution and the light detection region. Thus, it can be easily confirmed whether or not the measurement of the light intensity from can be performed.
- the determiner is configured such that when the magnitude of the light intensity detected by the photodetector deviates from the first light intensity reference value range, the signal intensity output by the photodetector. It may be configured to determine the reason why the measurement of the light intensity from the light detection region is not feasible based on the size of. According to such a configuration, when the measurement of the light intensity from the light detection region is not feasible, the reason is grasped, so that appropriate measures are taken so that the measurement of the light intensity from the light detection region can be performed. Is possible.
- the objective lens is If there is no liquid to be filled between the objective lens and the sample container in the case of the immersion type, (iii) if the light detection area is located on the wall of the sample container, and (iv) the light detector fails And (v) at least one of the cases where the excitation light is not focused on the light detection region in the state where the excitation light is required in the measurement of the light intensity.
- the range of the magnitude of the signal intensity output by the photodetector is stored in advance, and the magnitude of the signal intensity output by the photodetector is one of the previously stored ranges.
- Can measure the light intensity from the light detection area It is determined that there is no reason for the case corresponding to a prestored range to which the magnitude of the signal intensity output by the photodetector among the cases (i) to (v) belongs. It may be.
- Data in the range of the magnitude of the signal intensity stored in advance may be acquired by a preliminary experiment or the like performed in a state where the situation in the vicinity of the light detection region can be confirmed.
- the determiner determines the light intensity from the light detection region based on the magnitude of the signal intensity output by the light detector when the light detection region is not arranged in the sample solution. You may be comprised so that it may be determined whether there exists a cause which cannot perform measurement.
- the measurement of the light intensity from the photodetection region is performed while moving the position of the photodetection region in the sample solution.
- the photodetection area passes through the part deviating from the sample solution or the presence area of foreign matter, a change appears in the background of the signal intensity output from the photodetector, and the signal at that time The intensity typically increases.
- the movement path is a circulation path, a periodic increase in signal intensity is observed due to the presence of a portion where the light detection region deviates from the sample solution or foreign matter.
- the device of the present invention is a device that measures the light intensity from the light detection region while moving the position of the light detection region in the sample solution as in the scanning molecule counting method
- the first predetermined value and the second predetermined value may be set experimentally as appropriate, and may be the same value or different values.
- the determination by the determination unit may be automatically performed before the measurement of the light intensity from the light detection region.
- the light detection region is placed outside the sample container and output by a photodetector.
- the magnitude of the measured signal strength is referred to.
- the position of the light detection region is moved so as to cross the sample container, the magnitude of the signal intensity output by the photodetector at that time is sequentially referred to, and based on the magnitude of the signal intensity, The presence range of the wall, the range in the container, and / or the presence or absence of the sample solution may be confirmed.
- a warning may be issued to the user.
- the position of the light detection area is placed in the sample solution.
- the magnitude of the signal intensity that is moved on the planned path and output by the photodetector is referred to, and the magnitude of the signal intensity is compared with the first or second predetermined value, The presence or absence of a foreign substance or the like that deviates from the sample solution in the sample or affects the measurement of the light intensity may be confirmed. Then, when it is estimated that a part deviating from the sample solution or a foreign substance affecting the measurement of the light intensity is estimated on the movement path, the movement path may be changed.
- the light detection region is arranged in the sample solution and light detection is performed.
- the determination process that determines whether or not the light intensity from the area can be measured or the position or range of the light detection area relative to the sample container from which the light intensity can be measured can be realized by a general-purpose computer. It is. Therefore, according to another aspect of the present invention, the light intensity from the light detection region disposed in the sample solution is measured and analyzed using the optical system of the confocal microscope or the multiphoton microscope.
- the computer program is stored in a computer-readable storage medium and provided.
- the computer implements the above-described procedure by reading a program stored in a storage medium and executing information processing / arithmetic processing.
- the computer-readable recording medium may be a magnetic disk, a magneto-optical disk, a CD-ROM, a DVD-ROM, a semiconductor memory, or the like.
- the above-described program may be distributed to a computer via a communication line, and the computer that has received this distribution may execute the program.
- the magnitude of the signal intensity output by the photodetector is determined based on the intensity of the light from the light detection area where the pre-stored light detection area is arranged in the sample solution.
- the measurement of the light intensity from the light detection region can be performed at the position of the light detection region with respect to the sample container at that time It may be determined.
- the magnitude of the light intensity detected by the photodetector deviates from the first light intensity reference value range, based on the magnitude of the signal intensity output by the photodetector, The reason why the measurement of the light intensity is not feasible may be determined.
- the magnitude of the signal intensity output by the photodetector is stored in advance (i) when there is no sample solution in the sample container, and (ii) the objective lens is If there is no liquid to be filled between the objective lens and the sample container in the case of the immersion type, (iii) if the light detection region is located on the wall of the sample container, (iv) the light detector is When a failure occurs, or (v) when excitation light irradiation is necessary in the measurement of light intensity, the excitation light is not focused on the light detection area in the planned state.
- the reason why the measurement of the light intensity from the light detection area is not feasible when it is in one of the output signal intensity ranges is the light detection in the cases (i) to (v) Corresponds to a prestored range to which the magnitude of the signal intensity output by the device belongs. It may be determined. In the determination procedure, the reason why the light intensity from the light detection area cannot be measured based on the magnitude of the signal intensity output by the light detector when the light detection area is not arranged in the sample solution. It may be determined whether or not there is.
- the computer program is a computer program for light analysis for measuring the light intensity from the light detection region while moving the position of the light detection region in the sample solution.
- the position exceeding the first predetermined value in the magnitude of the signal intensity output by the photodetector while moving the position of the light detection area before executing the measurement of the light intensity from the light detection area is caused to execute a procedure for changing the movement path of the position of the light detection area. It may be.
- the determination procedure may be automatically executed by the computer before the measurement of the light intensity from the light detection region.
- the light detection region is arranged in the sample solution based on the magnitude of the signal intensity output by the light detector while moving the position of the light detection region with respect to the sample container.
- a novel method including a step of determining whether or not the measurement of the light intensity from the light detection region is feasible or the position of the light detection region with respect to the sample container or the range thereof where the light intensity measurement is feasible.
- an optical analysis method for analyzing the light intensity by measuring the light intensity from the light detection region arranged in the sample solution using an optical system of a confocal microscope or a multiphoton microscope.
- a movement process for moving the position of the light detection region relative to the sample container a signal detection process for detecting the signal intensity output by the photodetector while moving the position of the light detection area relative to the sample container, and the signal intensity. Based on the size of the light detection region, whether the light detection region is arranged in the sample solution and whether or not the light intensity from the light detection region can be measured, and the light detection for the sample container capable of performing the light intensity measurement
- a method includes a determination step of determining at least one of a region location or a range thereof.
- the magnitude of the signal intensity output by the photodetector is determined based on the intensity of the light from the light detection area.
- the measurement of the light intensity from the light detection region can be performed at the position of the light detection region with respect to the sample container at that time It may be determined.
- the magnitude of the light intensity detected by the photodetector deviates from the first light intensity reference value range, based on the magnitude of the signal intensity output by the photodetector, The reason why the measurement of the light intensity is not feasible may be determined.
- the magnitude of the signal intensity output by the photodetector is stored in advance (i) when there is no sample solution in the sample container, and (ii) the objective lens is If there is no liquid to be filled between the objective lens and the sample container in the case of the immersion type, (iii) if the light detection region is located on the wall of the sample container, (iv) the light detector is In case of failure or (v) when excitation light irradiation is necessary for light intensity measurement, output by the photodetector when the excitation light is not focused on the light detection area in the planned state
- the reason why the measurement of the light intensity from the light detection region is not feasible when it is in one of the signal intensity magnitude ranges is the photodetector in the cases (i) to (v) Corresponds to a pre-stored range to which the signal strength output by May be determined. Further, in the determination process, the light intensity from the light detection area cannot be measured based on the magnitude of the signal intensity output by the light detector
- the movement path of the position of the light detection area when applied to an optical analysis technique that measures the light intensity from the light detection region while moving the position of the light detection region in the sample solution, When there is a position that exceeds the first predetermined value in the magnitude of the signal intensity output by the photodetector while moving the position of the light detection area before performing the measurement of the light intensity from the detection area, or When there is a position that exceeds the second predetermined value in substantially the same period as the movement period of the position of the light detection area, the movement path of the position of the light detection area may be changed.
- the determination process may be automatically executed before the measurement of the light intensity from the light detection region.
- proteins, peptides, nucleic acids, lipids, sugars, and the like are processed according to the scanning molecule counting method (Patent Documents 6-8), FIDA (Patent Document 4), FCS (Patent Documents 1-3), Applied to optical analysis technology used for analysis or analysis of the state in solution of particulate biological objects such as chains, amino acids or aggregates thereof, viruses, cells, etc. It may be used to analyze or analyze the state of non-biological particles (eg, atoms, molecules, micelles, metal colloids, etc.) in solution, and such cases are also understood to be within the scope of the present invention. Should be.
- non-biological particles eg, atoms, molecules, micelles, metal colloids, etc.
- the optical intensity is output with reference to the magnitude of the signal intensity output from the photodetector. Since it is possible to determine whether the system is in a state where measurement of light intensity is feasible, actual inspections, etc., without depending on the user's experience or observation of the result state after measurement of light intensity It is possible to confirm whether or not the measurement can be performed correctly before performing the measurement.
- FIG. 1A is a schematic diagram of the internal structure of an optical analyzer that realizes the present invention.
- FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope).
- FIG. 1C is a schematic diagram of a mechanism for moving the position of the light detection region by changing the direction of the mirror 7.
- FIG. 1D is a schematic diagram of a mechanism for moving the position of the light detection region by moving the horizontal position of the microplate.
- FIG. 2A is a perspective view of a microplate used in the optical analyzer.
- FIG. 2B is a schematic diagram showing how the position of the objective lens moves relative to the microplate below the microplate.
- FIG. 3A is a schematic diagram of a microtube-type container having a flat bottom surface.
- FIG. 3B is a schematic side view when the light intensity is measured using a microtube type container in the optical analyzer.
- FIG. 3C shows a signal output from the photodetector when the confocal volume is moved in the horizontal direction at a pitch of 0.05 mm with respect to the microtube-type container in the state of FIG. It is the graph which showed intensity
- the left is when the sample solution is in the container, and the right is when the sample solution is not in the container.
- FIG. 4A is a diagram schematically showing a movement path when moving the position of the confocal volume in the sample solution. The left is a circular movement path, and the right is a linear movement path.
- FIG. 4B is a diagram schematically showing how the path is changed when the foreign object X exists on the movement path of the confocal volume.
- FIG. 4C shows a measurement example in the case where a foreign substance exists on the path in the measurement by the scanning molecule counting method.
- FIG. 5 is a diagram showing, in the form of a flowchart, one example of determination processing executed before the measurement of light intensity according to the present invention.
- Optical analyzer (confocal microscope) DESCRIPTION OF SYMBOLS 2 ... Light source 3 ... Single mode optical fiber 3a ... Fiber exit end 4 ... Collimator lens 4a ... Pinhole 5 ... Dichroic mirror 6, 7, 11 ... Reflection mirror 7a ... Mirror deflector 8 ... Objective lens 8a ... Immersion liquid 9 ... Microplate 9a ... Microtube type container 10 ... Well (sample solution container) 10a ... Well wall 10b ... Microplate bottom 12 ... Condenser lens 13 ... Pinhole 14 ... Barrier filter 15 ... Multimode optical fiber 16 ... Photo detector 17 ... Mirror deflector motor 17a ... Stage position changing device 18 ... Computer CV ... Confocal volume (light detection area)
- an optical analyzer 1 includes optical systems 2 to 17 and a computer 18 for controlling the operation of each part of the optical system and acquiring and analyzing data.
- the optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber.
- the light is emitted as a divergent light at an angle determined by a specific NA at the outgoing end of the light, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, the reflection mirrors 6, 7, and the objective lens 8. Is incident on.
- a microplate 9 in which a sample container or well 10 into which a sample solution of 1 to several tens of ⁇ L is dispensed is typically arranged is emitted from the objective lens 8.
- the laser light is focused in the sample solution in the sample container or well 10 to form a region with high light intensity (excitation region).
- single particles which are observation objects, typically luminescent particles to which luminescent labels such as fluorescent luminescent particles or fluorescent dyes are added are dispersed or dissolved, and these luminescent particles are excited.
- the luminescent particles When entering the region, the luminescent particles are excited and light is emitted.
- the emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter 14. (Here, only the light component of a specific wavelength band is selected.), Introduced into the multimode fiber 15, reaches the photodetector 16, is converted into a time-series electrical signal, and then to the computer 18. Input and processing for optical analysis.
- the pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, and as a result, as shown in FIG. Only the light emitted from the focal region of the laser beam, that is, the excitation region as schematically shown, passes through the pinhole 13, and the light from other than the excitation region is blocked.
- the focal region of the laser beam illustrated in FIG. 1B is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL (typically, the light intensity is in the region).
- the photodetector 16 is preferably an ultra-high light that can be used for photon counting.
- a sensitive photodetector is used.
- the time-series light intensity data is time-series photon count data.
- a stage position changing device 17a for moving the horizontal position of the microplate 9 may be provided on a microscope stage (not shown) in order to change the well 10 to be observed.
- the operation of the stage position changing device 17a may be controlled by the computer 18. With this configuration, it is possible to achieve quick measurement even when there are a plurality of specimens.
- a mechanism for scanning the sample solution with the light detection region that is, for moving the focus region, that is, the position of the light detection region in the sample solution.
- a mirror deflector 17 that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG. Change and move the absolute position of the light detection area).
- Such a mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope. Further, as illustrated in FIG.
- the horizontal position of the container 10 (microplate 9) into which the sample solution is injected is moved, and the relative position of the light detection region in the sample solution is shifted.
- the stage position changing device 17a is actuated to move the target position (method of moving the position of the sample solution).
- the mirror deflector 17 or the stage position changing device 17a cooperates with the light detection by the light detector 16 under the control of the computer 18 in order to achieve a desired movement pattern of the position of the light detection region.
- the movement trajectory of the position of the light detection region may be arbitrarily selected from a circle, an ellipse, a rectangle, a straight line, a curve, or a combination thereof (so that various movement patterns can be selected by the program in the computer 18). It may be.)
- the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 or the stage up and down.
- the above optical system is used as a multiphoton microscope. In that case, since there is light emission only in the focal region (light detection region) of the excitation light, the pinhole 13 may be removed. Further, when the luminescent particles to be observed emit light regardless of excitation light due to chemiluminescence or bioluminescence phenomenon, the optical systems 2 to 5 for generating excitation light may be omitted. When the luminescent particles emit light by phosphorescence or scattering, the optical system of the confocal microscope is used as it is.
- a plurality of excitation light sources 2 may be provided, and the wavelength of the excitation light may be appropriately selected according to the excitation wavelength of the luminescent particles.
- a dichroic mirror 14a may be inserted in the detection light path so that the detection light is divided into a plurality of wavelength bands and separately detected by a plurality of photodetectors 16.
- the present invention may be applied to an optical analysis technique of a type that detects a decrease in light intensity value due to the observation target particles entering the light detection region in the presence of significant background light.
- the optical system is the same as described above, but a luminescent substance that emits background light and observation target particles with relatively low emission intensity are dispersed in the sample solution.
- the computer 18 includes a CPU and a memory, and the CPU executes various arithmetic processes to execute the procedure of the present invention. Each procedure may be configured by hardware. All or a part of the processing described in the present embodiment may be executed by the computer 18 using a computer-readable storage medium storing a program for realizing the processing. That is, the computer 18 may realize the processing procedure of the present invention by reading a program stored in a storage medium and executing information processing / calculation processing.
- the computer-readable recording medium may be a magnetic disk, a magneto-optical disk, a CD-ROM, a DVD-ROM, a semiconductor memory, or the like.
- the above-mentioned program is distributed to a computer via a communication line. The computer that has received the distribution may execute the program.
- the light detection region does not exist in the sample solution ( The position of the light detection region is shifted from the sample solution, or the sample solution is not contained in the sample container.)
- the excitation light is necessary, the condensed state of the excitation light is abnormal, or If the measurement of the light intensity from the light detection region is executed without noticing that the output of the photodetector is abnormal, the measurement of the light intensity is wasted. Therefore, when measuring the light intensity, the optical system from the sample solution to the photodetector is in a state scheduled for measuring the light intensity from the light detection region, and the light intensity can be measured.
- the dimensions of the tip region of the objective lens, the sample container, and the like are relatively small, and it may be difficult for the user to visually confirm.
- a mechanism for observing the field of view of the objective lens such as an eyepiece lens or a camera for taking a microscopic image In such a case, it is more difficult to confirm the state of the vicinity of the light detection region in front of the objective lens in the optical axis direction.
- the magnitude of the signal intensity output from the photodetector is referred to while moving the position of the light detection region with respect to the sample container. Based on the size, whether or not a state in which the light detection region is arranged in the sample solution and measurement of the light intensity from the light detection region is feasible is achieved, or a sample in which such a state is achieved The position or range of the light detection area relative to the container is determined.
- FIG. 2 schematically shows the state of the determination process as described above in the present invention.
- the sample container is a microplate 9 in which a plurality of wells 10 are aligned as schematically illustrated in FIG. 2A, and the objective lens 8 is an immersion objective lens.
- the objective lens 8 is an immersion objective lens.
- there is a liquid between the objective lens 8 and the bottom surface 10b of the microplate 9 as schematically shown in FIG. 8a is provided, and the light detection region CV exists in the sample solution S.
- the positions of the well 10 and the objective lens 8 are shifted, and the light detection region CV is in the wall 10a of the well 10 (left figure) or the liquid 8a.
- the intensity of the scattered light or reflected light of the excitation light is measured in the light detection region CV in the sample solution S. Unlike the situation where is feasible, this difference appears in the signal intensity output by the photodetector. This also applies to the case where the excitation light Ex is not irradiated in the intended manner (when the excitation light is not irradiated or the condensing region is not located at the conjugate position of the objective lens / pinhole). Is reflected in the signal intensity output by the photodetector.
- the position of the objective lens 8 relative to the sample container is moved relative to the position of the light detection region relative to the sample container as indicated by the arrow in FIG.
- the signal intensity output by the photodetector is detected.
- the computer 18 stores in advance a signal intensity output by the photodetector in a state where the measurement of the light intensity is executable in a storage device, and moves the position of the objective lens 8 relative to the sample container.
- the output signal intensity of the photodetector detected at the time is compared with the signal intensity stored in advance, so that the light intensity can be measured when the light detection area is at an arbitrary position. It is possible to detect whether or not there is a position range where light intensity can be measured.
- each of the signal intensities output by the photodetector in a state where the measurement of the light intensity is not executable due to the various factors listed above is stored in advance in the storage device, If measurement of light intensity is not feasible when the light detection region is at an arbitrary position, the signal intensity at that time substantially matches any of the previously stored signal intensities corresponding to various factors. By determining whether or not the light intensity measurement is possible, it is possible to determine the factor that makes it impossible to measure the light intensity.
- the signal intensity in the above-described series of states stored in advance can be measured in advance under the same conditions as when measuring the light intensity of the same optical analyzer. Should. That is, for each measurement condition, once the signal intensity in a series of states is measured and stored in such a manner that the state of the optical system from the sample container to the photodetector can be confirmed, the stored signal intensity is thereafter stored. Will be available.
- FIG. 3 shows an example in which a microtube-type container 9a as schematically shown in FIG. 3A is used as a sample container.
- a microtube 9a is formed by flatly molding the bottom of a plastic microtube often used in this field, for example, in PCR.
- FIG. 3B a plurality of microtubes 9a are attached to a plate-shaped adapter having an opening, and the bottom surface of the microtube 9a is attached.
- the objective lens 8 is approached through the immersion liquid.
- the position of the objective lens 8 relative to the sample container is moved relative to the sample container as in the case of FIG.
- FIG. 3C shows the relative position of the objective lens 8 (water immersion objective lens) with respect to the sample container by 0.05 mm while checking the position of the light detection region, as illustrated in FIG. 3B.
- This shows an example of the output of the photodetector when it is moved.
- the vertical axis represents the output signal intensity of the photodetector, and is expressed in units [kHz] of the number of photons detected per measurement unit time (BIN TIME) (excitation light).
- the left plot is the result obtained in the container into which the sample solution was injected
- the right plot is the result obtained in the empty container.
- the output signal intensity of the photodetector is determined when the light detection area is outside the container, the wall of the container, the container where the sample solution exists, and the empty container. It will be understood that it varies with the structure of the container. More specifically, under the experimental conditions shown, the output signal intensity of the photodetector was as follows. (A) State in which the light detection region is outside the container (a-1) State in which there is no immersion water at the tip of the objective lens ...
- the position of the objective lens 8 relative to the sample container is moved relative to the sample container prior to the execution of the measurement.
- the output signal strength is detected.
- the detected signal intensity is a value when the measurement of the light intensity stored in advance can be executed [(c-1) a value when the light detection region is inside the container into which the sample solution is injected. ], It can be determined whether or not the measurement of the light intensity can be performed when the light detection region is at an arbitrary position.
- a light detection region in which the signal intensity output from the light detector substantially coincides with a value in the case where measurement of the light intensity stored in advance can be performed (within an allowable error range (the same applies hereinafter)).
- the position range By specifying the position range, it is possible to determine the position range where the light intensity measurement can be performed. Furthermore, since the signal intensity in the various states as described above (measured in such a manner that the state of the light detection region can be confirmed) is stored in the computer 18 in advance, the light intensity cannot be measured. Can be measured by determining whether the signal intensity output by the photodetector substantially matches or is close to the value stored in advance. The cause that is not can be identified.
- the position or range where the light intensity measurement determined as described above can be performed is stored in the storage device of the computer 18 in association with the configuration of the optical analyzer and the dimensions of the sample container, and is executed thereafter. It may be used when measuring the light intensity.
- Measurement of the light intensity from the light detection region CV is performed while scanning the sample solution with the light detection region CV as exemplified in 4 (A). In that case, if there are parts that deviate from the sample solution or foreign substances (X in FIG. 4B) on the movement path of the light detection region, the labor and labor to exclude those parts in the measurement data Is required. Therefore, in the aspect in which the light intensity is measured while scanning the sample solution with the light detection region, there is no part or foreign substance deviating from the sample solution on the movement path of the light detection region before the measurement. It is preferable to confirm this.
- the sample solution is obtained by referring to the output signal intensity of the moving light detector on the moving path of the light detection region, as in the case of determining whether the measurement of the light intensity is feasible. Therefore, it is possible to determine the presence or absence of a part or a foreign object that deviates from
- FIG. 4C shows the case where the light intensity is measured while moving the position of the light detection area, and the light intensity (photon count) when a foreign substance is present on the movement path of the light detection area.
- laser light having a wavelength of 640 nm was used as excitation light
- the output intensity of the objective lens was adjusted to 1 mW
- the light detection area was rotated and moved to 9000 rpm (period: 6667 ⁇ sec).
- the bin time was 10 ⁇ sec.
- a peak having a photon count exceeding 50 counts appeared at an interval substantially matching the moving period 6667 ⁇ sec of the light detection region.
- the light intensity emitted by the luminescent particles (ATTO647N, ATTO633, etc.) as the observation target particles is about 10 counts, so the peak of the photon count generated in synchronization with the movement period of the photodetection region in the figure is It is considered to be a foreign object that is fixedly present on the route.
- the movement path of the light detection region crosses the wall of the container, for example, as is understood from the result of FIG. .
- the position of the light detection region is moved along the movement path, and the output signal intensity of the light detector is detected.
- the detected output signal intensity has a value that exceeds an appropriately set threshold value, it may be determined that a portion deviating from the sample solution, a foreign substance, or the like exists on the movement path.
- the output signal intensity of the light detector obtained by moving the position of the light detection area along the movement path a plurality of times is set to the movement period of the light detection area.
- route may be suitably changed like FIG.4 (B) right figure.
- the above determination process in the present invention is a program (light for the sample container) stored in a storage device (not shown) of the computer 18 prior to execution of light intensity measurement after the sample container is installed. Whether the light detection area is arranged in the sample solution and the light intensity from the light detection area can be measured based on the magnitude of the signal intensity output by the photodetector while moving the position of the detection area Or a determination procedure for determining the position or range of the light detection region with respect to the sample container where light intensity can be measured, or a procedure for changing the movement path of the position of the light detection region) (i.e., The determination unit for executing the determination process is realized by an operation according to a program in the computer of the optical analyzer.) FIG.
- FIG. 5 shows one example of the determination process when a microplate having a plurality of wells is used or when a plurality of microtubes carried on an adapter as shown in FIG. 3 is used as a sample container.
- main measurement the measurement of the light intensity from the light detection region.
- the focus area (light detection area) of the objective lens 8 is changed.
- the stage on which the sample container is placed is moved so as to be positioned outside the sample container (step 100).
- the mirror deflector 7a below the objective lens 8 may be operated to move the photodetection region to the outside of the sample container.
- the output signal intensity of the photodetector is detected (step 110), and it is determined whether or not the detected output signal intensity is within a pre-stored threshold value Io ⁇ ⁇ o (step 120). ).
- the threshold Io is set to the signal intensity to be obtained in this state.
- step 120 when the detected output signal strength deviates from the range of the threshold value Io ⁇ ⁇ o, there is some abnormality, so that the detected output signal strength is stored in advance.
- the cause of the abnormality may be identified by comparing with the signal intensity value at the time of abnormality (step 125). Specifically, for example, the following treatment may be performed according to the value of the output signal strength.
- output signal intensity [value when there is no immersion liquid] (example: 0.3 kHz)
- a message indicating that there is no possibility of immersion is displayed, and the process is terminated.
- the apparatus has an automatic water supply mechanism, automatic water supply is performed and the determination process is executed again.
- step 120 when the detected output signal intensity is within the range of the threshold value Io ⁇ ⁇ o, the focal region of the objective lens 8 is positioned within the wall of the sample container according to the dimension information of the sample container.
- the stage on which the sample container is placed is moved (step 130), the output signal intensity of the photodetector is detected (step 140), and whether or not the detected output signal intensity is equal to or greater than a threshold Iw stored in advance.
- the detected output signal intensity does not exceed the threshold value Iw, it is estimated that the well or tube does not exist or the position of the well or tube is deviated from the planned position. May be displayed to skip the well or tube and proceed to the next well or tube, or allow the user to select whether to end the process (step 155).
- step 150 when the detected output signal intensity is equal to or greater than the threshold value Iw, the focal region of the objective lens 8 is positioned inside the sample container (well or tube) according to the dimension information of the sample container.
- the stage on which the sample container is placed is moved (step 160), the output signal intensity of the photodetector is detected (step 170), and the detected output signal intensity is within a pre-stored threshold value Ii ⁇ ⁇ i. It is determined whether or not there is (step 180). Since the state planned here is a state in which the focal region of the objective lens 8 is in the sample solution in the well of the sample container or the tube, the threshold value Ii is the signal intensity to be obtained in this state.
- threshold value Ii 0.7 kHz.
- ⁇ i is an error range that may be set as appropriate.
- the determination in step 180 may determine whether or not the output signal strength is equal to or greater than the threshold value Ii.
- the threshold value Ii is set corresponding to the background light of the sample solution used for the main measurement. For example, when the observation target particle is a light emitting particle having a low concentration, the solution that does not substantially emit light is a well or This is the signal strength when it is present in the tube. On the other hand, when a luminescent material that generates background light is dispersed in the sample solution, the threshold value Ii is the signal intensity when the solution in which the luminescent material is dispersed is present in the well or the tube.
- step 180 If it is determined in step 180 that the detected output signal intensity is not within the pre-stored threshold value Ii ⁇ ⁇ i (or less than the threshold value Ii), there is a sample solution in the well or tube. Or the position of the well or tube is presumed to deviate from the expected position, so this is indicated and the well or tube is skipped to the next well or tube. The user may be allowed to select whether to proceed or stop the processing (step 185).
- the light detection region is present in the sample solution. Thus, it is determined that the measurement of the light intensity is feasible (step 190). Note that the relative position of the focal region of the objective lens 8 with respect to the sample solution is moved, and the detected output signal intensity is within the threshold value Ii ⁇ ⁇ i stored in advance (or more than the threshold value Ii).
- a range of positions of the focal region of the lens 8, that is, a range of positions where the measurement of the light intensity can be performed may be defined.
- the light detection region in the sample solution is moved to a predetermined movement path.
- the output signal intensity of the photodetector is detected while moving along (step 200), and it is confirmed whether or not there is a part where the output signal intensity exceeds a predetermined threshold. In this process, if there is no portion where the output signal intensity exceeds a predetermined threshold, it is determined that the light intensity can be measured (step 220).
- treatment may be performed in the manner described below (step 225). Specifically, in the case where the position of the light detection region is moved along the circulation path (left in FIG. 4A: rotational scanning), a peak exceeding the threshold value appears regardless of the period of rotational scanning. When it appears, a message stating that there is a possibility that foreign matter may be mixed is displayed, and whether to execute the main measurement as it is, proceed to the next well or tube, or interrupt the processing It may be made to let a person choose.
- the center of the rotation scan is moved by an arbitrary distance (change of the movement path), and the rotation scan and the signal intensity are detected again, and the output signal intensity It is confirmed whether or not there is a part where the value exceeds a predetermined threshold. If a peak exceeding the threshold synchronized with the rotation cycle appears even if this process is executed a specified number of times, a message stating that there is a possibility that foreign matter is mixed in is displayed, and this measurement is performed as it is. The user may be allowed to choose between running, proceeding to the next well or tube, or interrupting the process.
- the light detection region is arranged in the sample solution before the execution of the measurement of the light intensity from the light detection region based on the signal intensity output by the photodetector.
- it is possible to confirm whether or not the state in which the measurement of the light intensity from the light detection region can be performed is achieved, it is caused by a defect in the state of the optical system from various sample solutions to the photodetector. Measurement failures will be prevented.
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Abstract
Description
2…光源
3…シングルモードオプティカルファイバー
3a…ファイバー出射端
4…コリメータレンズ
4a…ピンホール
5…ダイクロイックミラー
6、7、11…反射ミラー
7a…ミラー偏向器
8…対物レンズ
8a…液浸用液体
9…マイクロプレート
9a…マイクロチューブ型容器
10…ウェル(試料溶液容器)
10a…ウェルの壁部
10b…マイクロプレートの底部
12…コンデンサーレンズ
13…ピンホール
14…バリアフィルター
15…マルチモードオプティカルファイバー
16…光検出器
17…ミラー偏向器モーター
17a…ステージ位置変更装置
18…コンピュータ
CV…コンフォーカル・ボリューム(光検出領域)
本発明は、図1(A)に模式的に例示されている如き、走査分子計数法、FCS、FIDA、PCH等が実行可能な共焦点顕微鏡の光学系と光検出器とを組み合わせてなる光分析装置に適用される。図1(A)を参照して、光分析装置1は、光学系2~17と、光学系の各部の作動を制御すると共にデータを取得し解析するためのコンピュータ18とから構成される。光分析装置1の光学系は、通常の共焦点顕微鏡の光学系と同様であってよく、そこに於いて、光源2から放射されシングルモードファイバー3内を伝播したレーザー光(Ex)が、ファイバーの出射端に於いて固有のNAにて決まった角度にて発散する光となって放射され、コリメーター4によって平行光となり、ダイクロイックミラー5、反射ミラー6、7にて反射され、対物レンズ8へ入射される。対物レンズ8の上方には、典型的には、1~数十μLの試料溶液が分注される試料容器又はウェル10が配列されたマイクロプレート9が配置されており、対物レンズ8から出射したレーザー光は、試料容器又はウェル10内の試料溶液中で焦点を結び、光強度の強い領域(励起領域)が形成される。試料溶液中には、観測対象物である単一粒子、典型的には、蛍光性発光粒子又は蛍光色素等の発光標識が付加された発光粒子が分散又は溶解されており、かかる発光粒子が励起領域に進入すると、その間、発光粒子が励起され光が放出される。放出された光(Em)は、対物レンズ8、ダイクロイックミラー5を通過し、ミラー11にて反射してコンデンサーレンズ12にて集光され、ピンホール13を通過し、バリアフィルター14を透過して(ここで、特定の波長帯域の光成分のみが選択される。)、マルチモードファイバー15に導入されて、光検出器16に到達し、時系列の電気信号に変換された後、コンピュータ18へ入力され、光分析のための処理が為される。なお、当業者に於いて知られている如く、上記の構成に於いて、ピンホール13は、対物レンズ8の焦点位置と共役の位置に配置されており、これにより、図1(B)に模式的に示されている如きレーザー光の焦点領域、即ち、励起領域内から発せられた光のみがピンホール13を通過し、励起領域以外からの光は遮断される。図1(B)に例示されたレーザー光の焦点領域は、通常、1~10fL程度の実効体積を有する本光分析装置に於ける光検出領域であり(典型的には、光強度が領域の中心を頂点とするガウス様分布となる。実効体積は、光強度が1/e2となる面を境界とする略楕円球体の体積である。)、コンフォーカル・ボリュームと称される。また、本発明では、1つの発光粒子からの光、例えば、一つの蛍光色素分子からの微弱光が検出されるので、光検出器16としては、好適には、フォトンカウンティングに使用可能な超高感度の光検出器が用いられる。光の検出がフォトンカウンティングによる場合、光強度の測定は、所定時間に亘って、逐次的に、所定の単位時間毎(BIN TIME)に、光検出器に到来するフォトンの数を計測する態様にて実行される。従って、この場合、時系列の光強度のデータは、時系列のフォトンカウントデータである。また、顕微鏡のステージ(図示せず)には、観察するべきウェル10を変更するべく、マイクロプレート9の水平方向位置を移動するためのステージ位置変更装置17aが設けられていてよい。ステージ位置変更装置17aの作動は、コンピュータ18により制御されてよい。かかる構成により、検体が複数在る場合にも、迅速な計測が達成可能となる。
「発明の概要」にて触れたように、上記の光分析装置に於いて、光検出領域が試料溶液内に存在していないこと(光検出領域の位置が試料溶液からずれている、或いは、試料容器内に試料溶液が入っていないなど。)、励起光が必要な場合に励起光の集光状態に異常があること、或いは、光検出器の出力に異常があることなどに気付かずに、光検出領域からの光強度の計測を実行してしまった場合、かかる光強度の計測は無駄となってしまう。従って、光強度の計測の際には、試料溶液から光検出器までの光学系が光検出領域からの光強度の計測のために予定された状態に成っており、光強度の計測が実行可能であることを確認しておくことが望ましい。しかしながら、上記の光分析装置に於いて、対物レンズの先端領域、試料容器等の寸法は、比較的小さく、使用者が目視で確認することが困難であり得る。また、共焦点顕微鏡又は多光子顕微鏡がイメージングを目的とせず、光強度の計測を主要な目的としている場合、対物レンズの視野を観察するための機構、例えば、接眼レンズや顕微鏡像撮影用カメラ等、が設けられていないことがあり、その場合には、対物レンズの光軸方向前方の光検出領域の近傍の状態を確認することは一層困難である。
(i)励起光が照射されていない場合
(ii)光検出器が故障している場合
(iii)液浸式対物レンズの場合に対応する液体(水、シリコンオイル等)が対物レンズ先端に載置されていない場合[ドライ式対物レンズの場合、対物レンズ先端に異物が存在する場合]
(iv)光検出領域が容器の壁部に位置している場合
(v)ウェル内に試料溶液が注入されていない場合
(vi)その他の場合
(a)光検出領域が容器の外側に在る状態
(a-1)対物レンズの先端に液浸用水が無い状態 … 0.3kHz
(a-2)対物レンズの先端に液浸用水が在る状態 … 0.5kHz
(b)光検出領域が容器の壁部に在る状態 … 3.0kHz以上
(c)光検出領域が容器の内側に在る状態
(c-1)容器内に溶液が存在する場合 … 0.7kHz
(c-2)容器内が空の場合 … 0.2kHz
また、励起光が照射されていない場合は、0.2kHzであり、光検出器をOFFにしている場合は、0kHzであった。なお、上記の信号強度の値は、励起光強度に依存するところ、励起光強度が変化しても各々の大小関係は保存されることは確認された。上記の結果は、光検出器の出力信号強度を参照することにより、光検出領域が如何なる状態にあり、光強度の計測が実行可能であるか否かを判定でき、更に、光強度の計測が実行可能でない場合には、その原因を特定可能であることを示している。
ところで、既に触れた如く、走査分子計数法、或いは、FCS、FIDA、PCH等の幾つかの態様に於いては、図4(A)に例示されている如く試料溶液内を光検出領域CVにより走査しながら、光検出領域CVからの光強度の計測が実行される。その場合、光検出領域の移動経路上に於いて、試料溶液から逸脱する部分や異物(図4(B)中のX)が存在すると、計測データに於いてそれらの部分を除外する手間・労力が必要となる。従って、試料溶液内を光検出領域により走査しながら光強度の計測を行う態様に於いては、計測前に、光検出領域の移動経路上に試料溶液から逸脱する部分や異物が存在していないことを確認しておくことが好ましい。
本発明に於ける上記の判定処理は、試料容器の設置後に、光強度の計測実行に先立って、コンピュータ18の記憶装置(図示せず)に記憶されたプログラム(試料容器に対する光検出領域の位置を移動させながら光検出器により出力された信号強度の大きさに基づいて、光検出領域が試料溶液中に配置され且つ光検出領域からの光強度の計測が実行可能であるか否か又は光強度の計測が実行可能である試料容器に対する光検出領域の位置又はその範囲を判定する判定手順、光検出領域の位置の移動経路を変更する手順)に従って実行されてよい(即ち、判定処理を実行する判定器は、光分析装置のコンピュータに於けるプログラムに従った作動により実現される。)。図5は、複数のウェルを有するマイクロプレートを用いた場合又は図3の如きアダプタに担持された複数のマイクロチューブを試料容器として用いた場合の判定処理の過程の一つの例を示している。(以下、光検出領域からの光強度の計測を「本計測」と称する。)
(i)出力信号強度=[液浸用液体が無い場合の値]のとき(例:0.3kHz)
液浸水が無い可能性有り、の趣旨のメッセージを表示して、処理を終了させる。装置に自動給水機構がある場合は、自動給水を実施して再度判定処理を実行する。
(ii)出力信号強度=[光検出器のバックグラウンド値]のとき(例:0.2kHz)
レーザーが故障している可能性有り、の趣旨のメッセージを表示して、処理を終了させる。
(iii)出力信号強度<[光検出器のバックグラウンド値]のとき
光検出器が故障している可能性有り、の趣旨のメッセージを表示して、処理を終了させる。
Claims (21)
- 共焦点顕微鏡又は多光子顕微鏡の光学系を用いて試料溶液中に配置された光検出領域からの光強度を計測し前記光強度の分析を行う光分析装置であって、
試料容器に対する前記光検出領域の位置を移動する光検出領域位置移動器と、
前記光検出領域からの光を検出するための光検出器と、
前記試料容器に対する前記光検出領域の位置を移動させながら前記光検出器により出力された信号強度の大きさに基づいて、前記光検出領域が前記試料溶液中に配置され且つ前記光検出領域からの光強度の計測が実行可能であるか否か及び前記光強度の計測が実行可能である前記試料容器に対する前記光検出領域の位置又はその範囲のうちの少なくとも一つを判定する判定器と
を含むことを特徴とする装置。 - 請求項1の装置であって、前記判定器が、前記光検出領域が前記試料溶液中に配置され且つ前記光検出領域からの光強度の計測が実行可能であるときの第一の信号強度参照値範囲を記憶しており、前記光検出器により出力された信号強度の大きさが前記第一の光強度参照値範囲に在るとき、そのときの前記試料容器に対する前記光検出領域の位置に於いて前記光検出領域からの光強度の計測が実行可能であると判定することを特徴とする装置。
- 請求項2の装置であって、前記光検出器により検出された光強度の大きさが前記第一の光強度参照値範囲から逸脱しているとき、前記光検出器により出力された信号強度の大きさに基づいて、前記光検出領域からの光強度の計測が実行可能でない理由が判定されることを特徴とする装置。
- 請求項3の装置であって、(i)前記試料容器内に試料溶液がない場合、(ii)対物レンズが液浸式である場合に前記対物レンズと前記試料容器との間に満たされるべき液体がない場合、(iii)前記光検出領域が前記試料容器の壁に位置している場合、(iv)前記光検出器が故障している場合及び(v)前記光強度の計測に於いて励起光の照射が必要な場合に前記励起光が予定された状態にて前記光検出領域に集光されていない場合のうちの少なくとも一つの場合の前記光検出器により出力される信号強度の大きさの範囲が予め記憶され、前記光検出器により出力される信号強度の大きさが前記予め記憶された範囲のうちの一つに在るとき、前記光検出領域からの光強度の計測が実行可能でない理由が、前記(i)~(v)の場合のうちの前記光検出器により出力される信号強度の大きさの属する前記予め記憶された範囲に対応する場合であることが判定されることを特徴とする装置。
- 請求項1又は2の装置であって、前記判定器が、前記光検出領域が前記試料溶液内に配置されていないときに前記光検出器により出力された信号強度の大きさに基づいて前記光検出領域からの光強度の計測が実行できない原因があるか否かを判定することを特徴とする装置。
- 請求項1乃至5のいずれかの装置であって、前記試料溶液内に於ける前記光検出領域の位置を移動しながら前記光検出領域からの光強度の計測を行う装置にして、前記光検出領域からの光強度の計測の実行の前に前記光検出領域の位置を移動しながら前記光検出器により出力された信号強度の大きさに於いて第一の所定値を超える位置があったとき又は前記光検出領域の位置の移動周期と略同じ周期にて第二の所定値を超える位置があったときには、前記光検出領域の位置の移動経路を変更することを特徴とする装置。
- 請求項1乃至6のいずれかの装置であって、前記判定器による前記判定が前記光検出領域からの光強度の計測の実行前に自動的に為されることを特徴とする装置。
- 共焦点顕微鏡又は多光子顕微鏡の光学系を用いて試料溶液中に配置された光検出領域からの光強度を計測し前記光強度の分析を行う光分析方法であって、
試料容器に対する前記光検出領域の位置を移動させる移動過程と、
前記試料容器に対する前記光検出領域の位置を移動させながら光検出器により出力された信号強度を検出する信号検出過程と、
前記信号強度の大きさに基づいて、前記光検出領域が前記試料溶液中に配置され且つ前記光検出領域からの光強度の計測が実行可能であるか否か及び前記光強度の計測が実行可能である前記試料容器に対する前記光検出領域の位置又はその範囲のうちの少なくとも一つを判定する判定過程と
を含むことを特徴とする方法。 - 請求項8の方法であって、前記判定過程に於いて、前記光検出器により出力された信号強度の大きさが、予め記憶された前記光検出領域が前記試料溶液中に配置され且つ前記光検出領域からの光強度の計測が実行可能であるときの第一の信号強度参照値範囲に在るとき、そのときの前記試料容器に対する前記光検出領域の位置に於いて前記光検出領域からの光強度の計測が実行可能であると判定されることを特徴とする方法。
- 請求項9の方法であって、前記光検出器により検出された光強度の大きさが前記第一の光強度参照値範囲から逸脱しているとき、前記光検出器により出力された信号強度の大きさに基づいて、前記光検出領域からの光強度の計測が実行可能でない理由が判定されることを特徴とする方法。
- 請求項10の方法であって、前記光検出器により出力される信号強度の大きさが、予め記憶された(i)前記試料容器内に試料溶液がない場合、(ii)対物レンズが液浸式である場合に前記対物レンズと前記試料容器との間に満たされるべき液体がない場合、(iii)前記光検出領域が前記試料容器の壁に位置している場合、(iv)前記光検出器が故障している場合、又は(v)前記光強度の計測に於いて励起光の照射が必要な場合に前記励起光が予定された状態にて前記光検出領域に集光されていない場合の前記光検出器により出力される信号強度の大きさの範囲のうちの一つに在るとき、前記光検出領域からの光強度の計測が実行可能でない理由が、前記(i)~(v)の場合のうちの前記光検出器により出力される信号強度の大きさの属する前記予め記憶された範囲に対応する場合であることが判定されることを特徴とする方法。
- 請求項8又は9の方法であって、前記判定過程に於いて、前記光検出領域が前記試料溶液内に配置されていないときに前記光検出器により出力された信号強度の大きさに基づいて前記光検出領域からの光強度の計測が実行できない原因があるか否かが判定されることを特徴とする方法。
- 請求項8乃至12のいずれかの方法であって、前記試料溶液内に於ける前記光検出領域の位置を移動しながら前記光検出領域からの光強度の計測を行う方法にして、前記光検出領域からの光強度の計測の実行の前に前記光検出領域の位置を移動しながら前記光検出器により出力された信号強度の大きさに於いて第一の所定値を超える位置があったとき又は前記光検出領域の位置の移動周期と略同じ周期にて第二の所定値を超える位置があったときには、前記光検出領域の位置の移動経路を変更することを特徴とする方法。
- 請求項8乃至13のいずれかの方法であって、前記光検出領域からの光強度の計測の実行前に前記判定過程を自動的に実行することを特徴とする方法。
- 共焦点顕微鏡又は多光子顕微鏡の光学系を用いて試料溶液中に配置された光検出領域からの光強度を計測し前記光強度の分析を行うための光分析用コンピュータプログラムであって、
試料容器に対する前記光検出領域の位置を移動する手順と、
試料容器に対する前記光検出領域の位置を移動させながら光検出器により出力された信号強度を検出する手順と、
前記信号強度の大きさに基づいて、前記光検出領域が前記試料溶液中に配置され且つ前記光検出領域からの光強度の計測が実行可能であるか否か及び/又は前記光強度の計測が実行可能である前記試料容器に対する前記光検出領域の位置又はその範囲のうちの少なくとも一つを判定する判定手順と
をコンピュータに実行させることを特徴とするコンピュータプログラム。 - 請求項15のコンピュータプログラムであって、前記判定手順に於いて、前記光検出器により出力された信号強度の大きさが、予め記憶された前記光検出領域が前記試料溶液中に配置され且つ前記光検出領域からの光強度の計測が実行可能であるときの第一の信号強度参照値範囲に在るとき、そのときの前記試料容器に対する前記光検出領域の位置に於いて前記光検出領域からの光強度の計測が実行可能であると判定されることを特徴とするコンピュータプログラム。
- 請求項16のコンピュータプログラムであって、前記光検出器により検出された光強度の大きさが前記第一の光強度参照値範囲から逸脱しているとき、前記光検出器により出力された信号強度の大きさに基づいて、前記光検出領域からの光強度の計測が実行可能でない理由が判定されることを特徴とするコンピュータプログラム。
- 請求項17のコンピュータプログラムであって、前記判定手順に於いて、前記光検出器により出力される信号強度の大きさが、予め記憶された(i)前記試料容器内に試料溶液がない場合、(ii)対物レンズが液浸式である場合に前記対物レンズと前記試料容器との間に満たされるべき液体がない場合、(iii)前記光検出領域が前記試料容器の壁に位置している場合、(iv)前記光検出器が故障している場合、又は(v)前記光強度の計測に於いて励起光の照射が必要な場合に前記励起光が予定された状態にて前記光検出領域に集光されていない場合の前記光検出器により出力される信号強度の大きさの範囲のうちの一つに在るとき、前記光検出領域からの光強度の計測が実行可能でない理由が、前記(i)~(v)の場合のうちの前記光検出器により出力される信号強度の大きさの属する前記予め記憶された範囲に対応する場合であることが判定されることを特徴とするコンピュータプログラム。
- 請求項15又は16のコンピュータプログラムであって、前記判定手順に於いて、前記光検出領域が前記試料溶液内に配置されていないときに前記光検出器により出力された信号強度の大きさに基づいて前記光検出領域からの光強度の計測が実行できない原因があるか否かが判定されることを特徴とするコンピュータプログラム。
- 請求項15乃至19のいずれかのコンピュータプログラムであって、前記試料溶液内に於ける前記光検出領域の位置を移動しながら前記光検出領域からの光強度の計測を行うための光分析用コンピュータプログラムにして、前記光検出領域からの光強度の計測の実行の前に前記光検出領域の位置を移動しながら前記光検出器により出力された信号強度の大きさに於いて第一の所定値を超える位置があったとき又は前記光検出領域の位置の移動周期と略同じ周期にて第二の所定値を超える位置があったときには、前記光検出領域の位置の移動経路を変更する手順をコンピュータに実行させることを特徴とするコンピュータプログラム。
- 請求項15乃至20のいずれかのコンピュータプログラムであって、前記光検出領域からの光強度の計測の実行前に前記判定手順を自動的にコンピュータに実行させることを特徴とするコンピュータプログラム。
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WO2021171419A1 (ja) * | 2020-02-26 | 2021-09-02 | 株式会社日立ハイテク | 検体観察装置、検体観察方法 |
JP2022508329A (ja) * | 2018-12-07 | 2022-01-19 | ライカ マイクロシステムズ シーエムエス ゲゼルシャフト ミット ベシュレンクテル ハフツング | 試料配置部上の位置を自動的に特定するための方法および対応する顕微鏡 |
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