CN103776769A - Confocal optical scanner - Google Patents
Confocal optical scanner Download PDFInfo
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- CN103776769A CN103776769A CN201210398683.1A CN201210398683A CN103776769A CN 103776769 A CN103776769 A CN 103776769A CN 201210398683 A CN201210398683 A CN 201210398683A CN 103776769 A CN103776769 A CN 103776769A
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Abstract
The invention relates to a confocal optical scanner, and especially relates to a confocal optical scanner using array detectors comprising CCD, EMCC, CMOS and sCOMS as detection units and applied to biological fluorescent microscopic imaging. The confocal optical scanner is mainly characterized in that microlenses of a microlens array corresponds to pinholes of a pinhole array one by one, and the pinhole array is positioned in the focal plane of the microlenses; excitation light illuminating a sample and fluorescence emitted by the sample pass through a same pinhole in the pinhole array; the pinholes of the pinhole array are arranged in an equidistant matrix manner, and illumination points of the pinholes on the sample have a same moving speed; and a scan vibrating mirror has two reflection planes, and the scanning imaging of the sample is realized through the reciprocating rotation of the scan vibrating mirror. So the confocal optical scanner has the advantages of fast scanning speed, zero stray light background noise, uniform illumination intensity and the like, and can be easily synchronously controlled together with the exposure of the detectors.
Description
Technical field
The present invention relates to a kind of spininess hole confocal optical scanner, particularly use the face battle array detecting devices such as CCD, EMCCD, CMOS and sCOMS as spininess detecting unit, that be applied to micro-imaging hole confocal optical scanner.The present invention is mainly used in biomedical micro-imaging field, also can be used for investigation of materials and integrated circuit (IC) chip is detected as picture.
Background technology
Along with going deep into of RESEARCH ON CELL-BIOLOGY, the application of fluorescent microscope imaging is more and more general, and confocal microscopic image is paid attention to especially widely.But current confocal imaging system has variously cannot overcome problem, makes the performance of system be difficult to further raising:
1. the image taking speed of single needle hole laser co-focusing optical scanner is generally lower, and under typical 1024 × 1024 pixel image-forming conditions, image taking speed is less than 1 hertz; And single needle hole laser scanning co-focusing microscope use photomultiplier as detecting element, photoelectric transformation efficiency is not high, only has 30% all quantum efficiencies.
2. the pin hole that rotating disk confocal optical scanner utilizes the rotating disk of High Rotation Speed to drive isometric helix to arrange scans sample.Although the sweep velocity that it has 1000 hertz, the pin hole that is positioned at rotating disk internal diameter has slower rotational speed than the pin hole that is positioned at rotating disk external diameter, makes each pin hole different in size to the lighting hours of sample, causes illumination intensity inhomogeneous.And the rotating disk of High Rotation Speed adjusting rotary speed in real time makes the exposure of illumination to sample and detecting device can not Complete Synchronization, brings light and dark sweep trace to image, has reduced picture quality.
3. the slide block that the utilization of slide block confocal scanning instrument reciprocatingly slides is obtained multiple pin hole scanning samples, and because current technical limitation cannot realize high-velocity scanning, sweep velocity only has 20 hertz the soonest.
Summary of the invention
The weak point that the object of the invention is to exist for current various confocal optical scanners is improved, as slow in single-point confocal laser scanning speed of scanner, sensitivity is low; Rotating disk confocal scanning instrument has bright dark alternate sweep trace interference, illumination intensity heterogeneity; The problems such as slide block confocal scanning instrument sweep velocity is slow, provide a kind of new spininess hole confocal optical scanner, coordinate fluorescent microscope and face battle array detecting device composition confocal imaging system, thereby obtain at high speed high-quality Confocal Images.
The object of the invention is to realize by following technical scheme:
As shown in Figure 1, the invention provides a kind of spininess hole confocal optical scanner, mainly comprise: light source 1; Excite color filter 2; Illuminating lens group 3; Microlens array 4, has arranged lenticule 4a on it; Dichroic beam splitter 5; Pinhole array 6, has arranged pin hole 6a on it; Delay lens group 7; Scanning galvanometer 8, has the first reflecting surface 8a and the second reflecting surface 8b; Cut-off diaphragm 9, is divided into light transmission part 9a and lightproof part 9b; Total reflective mirror group 10; Imaging lens group 11; Transmitting color filter 12; Controller 13; It is characterized in that:
Described light source 1 use in laser, light emitting diode, mercury lamp, xenon lamp and metal halide lamp any one or multiple as light source;
The described effect that excites color filter 2 is, see through the part wavelength that described light source 1 launches light, reflect the light of other wavelength, obtain the exciting light for the sample that throws light on;
The effect of described illuminating lens group 3 is, make described light source 1 launch and see through described in excite the exciting light of color filter 2 to form parallel beam;
Described microlens array 4 is positioned at described illuminating lens group 3, and perpendicular to described parallel beam, has arranged described lenticule 4a multiple printing opacities, that have phase parfocal on it, and remainder is light tight; Described lenticule 4a is circle or polygon, as triangle, square, rectangle, quadrilateral, pentagon and hexagon etc.; Described lenticule is one or more in Fresnel lens, curved reflector, miniature lenticular lens type lenticule, and has equal focal length;
Described dichroic beam splitter 5, between described microlens array 4 and described pinhole array 6, has 45 degree angles with described parallel beam;
After described pinhole array 6 is placed on described dichroic beam splitter 5, perpendicular to described parallel beam, and be positioned on the focal plane of described dichroic beam splitter 5 one sides of described lenticule 4a;
The described pin hole 6a that has arranged multiple printing opacities on described pinhole array 6, remainder is light tight; Described pin hole 6a is circle or polygon, as triangle, square, rectangle, quadrilateral, pentagon and hexagon etc.;
When described pin hole 6a is circular, the big or small r of described pin hole 6a is defined as the radius of described circle; When described pin hole 6a is polygon, the big or small r of described pin hole 6a is defined as described polygonal inscribe radius of a circle;
The big or small r of described pin hole 6a is determined by the enlargement ratio M2 of numerical aperture NA, the enlargement ratio M1 of the object lens 15 of microscope 19, described delay lens group 7 and the wavelength X of described sample 16 emitting fluorescences:
r＝0.61×λ÷NA×M1×M2
As shown in Figure 8, the described pin hole 6a on described pinhole array 6 is according to regularly arranged below: in the time that described scanning galvanometer 8 rotates, described lighting point mobile direction on described sample 16 is defined as direction of scanning; The arrangement of the described pin hole 6a that is parallel to described direction of scanning is defined as to row; The arrangement of described pin hole 6a perpendicular to described direction of scanning is defined as to row; The quantity of the described pin hole 6a of every a line is equal, and the quantity of the described pin hole 6a of each row equates, and each walks to a rare described pin hole 6a; Draw and be parallel to the straight line of described direction of scanning through described pin hole 6a center, the distance of two adjacent straight lines is the line space of pin hole 6a described in adjacent two row, is called I; Draw the straight line perpendicular to direction of scanning through described pin hole 6a center, the distance of two adjacent straight lines is the column pitch of the described pin hole 6a of adjacent two row, is called J; Centre distance with adjacent two described pin hole 6a of a line is defined as K; The centre distance of adjacent two described pin hole 6a of same row is defined as L; The arrangement of described pin hole 6a makes the big or small r of I, J, K and L and described pin hole 6a have following equation relation:
0＜I≤2r
L＝n×I，
K
2＝n
2×L
2-I
2
J
2＝L
2-I
2
N is the coefficient of described pin hole 6a spacing for a change, 1 < n≤20;
As shown in Figure 9, described lenticule 4a and described pin hole 6a have identical arrangement, form one-to-one relationship through described dichroic beam splitter 5, described parallel beam by described lenticule 4a beam splitting, converge, the focus converging is positioned at the described pin hole 6a center after described dichroic beam splitter 5; Described parallel beam beam splitting forms multiple pointolites after seeing through described pin hole 6a;
Described delay lens group 7 is to described pinhole array 6 imagings, and the picture planes overlapping to described sample 16 imagings as plane and described object lens 15 forms and described pin hole 6a lighting point one to one described pointolite on described sample 16;
Described scanning galvanometer 8, around fixed rotating shaft reciprocating rotary, has two reflectings surface, i.e. the first reflecting surface 8a and the second reflecting surface 8b; The first reflecting surface 8a and the second reflecting surface 8b are plane reflection or concave reflection; In the time that described the first reflecting surface 8a and the second reflecting surface 8b are concave reflection, have and equate or focal length not etc.;
Described cut-off diaphragm 9 is positioned at the picture plane of described sample 16 through described object lens 15 imagings, is divided into described light transmission part 9a and described lightproof part 9b;
Described total reflective mirror group 10 is plane reflection or concave reflection;
Described imaging lens group 11, to described pinhole array 6 imagings, is positioned at as plane on the sensitive chip 14 of face battle array detecting device 20;
The effect of described transmitting color filter 12 is, sees through the fluorescence of the specific wavelength that described sample 16 launches, and reflects the light of other wavelength;
The effect of described controller 13 is: control position of rotation, the swing circle of described scanning galvanometer 8, and carry out signal and communication with described battle array detecting device 20, realize the rotation of described scanning galvanometer 8 and the exposure synchro control of described battle array detecting device 20.
Advantage of the present invention is as follows:
1, the present invention drives scanning by scanning galvanometer, is used in conjunction with the high sensitivity face battle array detecting devices such as CCD, EMCCD, CMOS and sCOMS, can realize high speed, the burnt optical imagery of highly sensitive copolymerization.
2, the present invention is single pinhole array, and exciting light and fluorescence, through same pinhole array, are installed and regulated simply.
3, the present invention uses microlens array and the two array structures of pinhole array, and lenticule and pin hole are corresponding one by one, and light source utilization factor is high, without parasitic light ground unrest.
4, the scanning of scanning galvanometer of the present invention is synchronizeed with the exposure of face battle array detecting device, the bright dark inhomogeneous problem of image of avoiding scanning not exclusively to cause.
5, the present invention is completely equal to the illumination intensity of sample, visual field illumination intensity homogeneous.
Accompanying drawing explanation
Fig. 1: the structural representation of the present invention and microscope and face battle array detector applies
Fig. 2 a and 2b: the schematic diagram of the first embodiment of the present invention
Fig. 3: the schematic diagram of the second embodiment of the present invention
Fig. 4: the schematic diagram of the third embodiment of the present invention
Fig. 5: the schematic diagram of the 4th kind of embodiment of the present invention
Fig. 6: the schematic diagram of the 5th kind of embodiment of the present invention
Fig. 7: the schematic diagram of the 6th kind of embodiment of the present invention
Fig. 8: the schematic diagram of pinhole array arrangement mode of the present invention
Fig. 9: the schematic diagram of circular pinhole array of the present invention and circular microlens array
Drawing explanation:
1-light source 2-excites color filter 3-illuminating lens group 4-microlens array 4a-lenticule 5-dichroic beam splitter 6-pinhole array 6a-pin hole 7-delay lens group 8-scanning galvanometer 8a-scanning galvanometer first reflecting surface 8b-scanning galvanometer the second reflecting surface 9-cut-off diaphragm 9a-light transmission part 9b-lightproof part 10-total reflective mirror group 11-imaging lens group 12-transmitting color filter 13-controller 14-sensitive chip 15-object lens 16-sample 17-computing machine 18-graphoscope 19-microscope 20-face battle array detecting device
Embodiment
Further describe the present invention below in conjunction with drawings and Examples.
Fig. 2 a and 2b are the schematic diagram of the first confocal optical scanner related to the present invention, and wherein, the first reflecting surface 8a of scanning galvanometer 8, the second reflecting surface 8b and total reflective mirror group 10 are all plane reflections.
As Fig. 2 a, in the present embodiment, face battle array detecting device 20 (not shown)s start before exposure, and controller 13 (not shown) gated sweep galvanometers 8 rest on maximum spin angular position forward or backwards; Light is launched from light source 1, through exciting the pin hole 6a on lenticule 4a, dichroic beam splitter 5 and the pinhole array 6 on color filter 2, illuminating lens group 3, microlens array 4 to form multiple shot point light sources; Delayed lens combination 7 imagings of shot point light source the first reflecting surface 8a that is scanned galvanometer 8 are reflected in the lightproof part 9b of cut-off diaphragm 9, can not enter microscope 19 (not shown)s, on the sample 16 that is positioned at object lens 15 focal planes, form lighting point.
As Fig. 2 b, battle array detecting device 20 (not shown)s start exposure face to face, controller 13 (not shown) gated sweep galvanometers 8 start clockwise or are rotated counterclockwise to the reverse or maximum spin angular position of forward from maximum spin angular position forward or backwards, make shot point light source be entered microscope 19 (not shown)s through the light transmission part 9a of cut-off diaphragm, on the sample 16 that is positioned at object lens 15 focal planes, form lighting point.Along with the rotation of scanning galvanometer 8, lighting point moves on sample 16.When battle array detecting device 20 (not shown) end exposure, scanning galvanometer 8 just rotate to reverse or the maximum spin angular position of forward face to face, and sample 16 is once illuminated.
The arrangement of all lighting points on sample 16 is identical with the arrangement of pin hole 6a, is all that isometric matrix is arranged, and moves on sample 16 with identical speed.So in 20 (not shown)s whens exposure of face battle array detecting device, sample 16 is by illumination is once intactly, equably; Or, by the swing circle of controller 13 (not shown) gated sweep galvanometers 8, make the time shutter of face battle array detecting device 20 (not shown)s equal the integral multiple of the swing circle of scanning galvanometer 8, sample 16 in the time of the 20 (not shown)s exposure of face battle array detecting device by repeatedly intactly, illumination equably.
The fluorescence that lighting point excited sample 16 is launched is reflected by dichroic beam splitter 5 through the pin hole 6a of the first reflecting surface 8a, delay lens group 7 and the pinhole array 6 of object lens 15, the light transmission part 9a that ends diaphragm 9, scanning galvanometer 8, then is formed into picture point through the second reflecting surface 8b and the transmitting color filter 12 of total reflective mirror group 10, imaging lens group 11, scanning galvanometer 8 at sensitive chip 14.Imaging point moves along with scanning galvanometer 8 is rotated on sensitive chip 14, in the time of face battle array detecting device 20 (not shown) end exposure, sensitive chip 14 obtains the complete fluorescence information of sample 16, and by computing machine 17 (not shown) processing, be shown as image in graphoscope 18 (not shown)s.Could pass through the pin hole 6a of pinhole array 6, reflected by dichroic beam splitter 5 and see through transmitting color filter 12 and arrive sensitive chip 14 owing to being only positioned at fluorescence that the sample 16 of focal plane of object lens 15 launches, so the image showing in graphoscope 18 (not shown)s is the Confocal Images of sample 16.
Fig. 3 is the structural representation of the second confocal optical scanner related to the present invention, specific as follows with the difference of embodiment 1: the first reflecting surface 8a, the second reflecting surface 8b of scanning galvanometer 8, and first total reflective mirror 10 between reflecting surface 8a and pinhole array 6 be all concave reflection, can distinguish the delay lens group 7 of alternate embodiment 1 and imaging lens group 11 to pinhole array 6 imagings, the imaging multiplying power of the total reflective mirror 10 between the first reflecting surface 8a and the first reflecting surface 8a and pinhole array 6 is M2.Because reduced delay lens group 7 and imaging lens group 11, in the light path of the present embodiment, there is optical interface still less, can obtain higher optical efficiency.
Fig. 4 is the structural representation of the third confocal optical scanner related to the present invention, specific as follows with the difference of embodiment 2: the first reflecting surface 8a of scanning galvanometer 8 and total reflective mirror group 10 for concave reflection, the second reflecting surface 8b be plane reflection.
Fig. 5 is the structural representation of the 4th kind of confocal optical scanner related to the present invention, specific as follows with the difference of embodiment 3: the first reflecting surface 8a and the second reflecting surface 8b, the total reflective mirror group 10 of scanning galvanometer 8 are all concave reflections.
Fig. 6 is the structural representation of the 5th kind of confocal optical scanner related to the present invention, specific as follows with the difference of embodiment 4: the first reflecting surface 8a and the second reflecting surface 8b are respectively on two identical scanning galvanometers 8, rotated by controller 13 synchro control, reduced a slice total reflective mirror 10.
Fig. 7 is the structural representation of the 6th kind of confocal optical scanner related to the present invention, specific as follows with the difference of embodiment 1: to have increased the quantity of imaging lens group 11, made image and object lens 15 that sensitive chip 14 records can realize amplification in strict 1: 1 to the imaging of sample 16.
Claims (10)
1. a confocal optical scanner, launch and see through the light of pinhole array by light source by there being the scanning galvanometer reflection of two reflectings surface, illumination is positioned at the sample of micro objective focal plane, and by the rotation of described scanning galvanometer, described sample is scanned, finally launch and see through the fluorescence of described pinhole array by sample described in face battle array detector recording, complete co-focusing imaging, it is characterized in that:
Have a described pinhole array, arranged the pin hole of multiple printing opacities on it, remainder is light tight;
The light beam of described light source transmitting is divided into multiple pointolites by described pin hole, and reflect through the first reflecting surface of described scanning galvanometer, on the described sample that is positioned at described micro objective focal plane, form multiple and described pin hole lighting point one to one, described sample throws light on simultaneously;
The fluorescence of described sample transmitting is after described micro objective, the first reflecting surface reflection by described scanning galvanometer sees through described pin hole, through the second reflecting surface reflection of described scanning galvanometer, on the sensitive chip of described battle array detecting device, form multiple picture point again;
The rotation of described scanning galvanometer, makes described lighting point move on described sample with identical speed, realize to described sample completely, illumination uniformly; Make described picture point move on the sensitive chip of described battle array detecting device simultaneously, obtain the fluoroscopic image of complete described sample, complete co-focusing imaging.
2. a kind of confocal optical scanner according to claim 1, between described light source and described pinhole array, also place one and arranged multiple lenticular microlens arrays, it is characterized in that: described pinhole array is positioned at described lenticular focal plane, and the described lenticule of described microlens array is corresponding one by one with the described pin hole of described pinhole array, be the light beam of described light source transmitting by the beam splitting of described lenticule battle array, converge, the focus converging is positioned at the center of described pin hole, then sees through described pinhole array and forms described pointolite.
3. a kind of confocal optical scanner according to claim 2, it is characterized in that: the described lenticule of described microlens array is one or more in Fresnel lens, curved reflector, miniature lenticular lens type lenticule, be shaped as circle or polygon, and all there is equal focal length.
4. a kind of confocal optical scanner according to claim 1 and 2, is characterized in that: described pin hole is circle or polygon; In the time that described pin hole is circle, the big or small r of described pin hole is defined as the radius of described circle; In the time that described pin hole is polygon, described pin hole big or small r be defined as described polygonal inscribe radius of a circle.
5. a kind of confocal optical scanner according to claim 4, it is characterized in that: the described pin hole on described pinhole array is according to regularly arranged below: in the time of described scanning galvanometer rotation, described lighting point mobile direction on described sample is defined as direction of scanning; The arrangement of the described pin hole that is parallel to described direction of scanning is defined as to row, the arrangement of the described pin hole perpendicular to described direction of scanning is defined as to row; The quantity of the described pin hole of every a line is equal, and the quantity of the described pin hole of each row equates, and each walks to a rare described pin hole; Draw the straight line that is parallel to described direction of scanning through the center of described pin hole, the distance of two adjacent straight lines is the line space of pin hole described in adjacent two row, is called I; The straight line perpendicular to described direction of scanning is drawn at center through described pin hole, and the distance of two adjacent straight lines is the column pitch of the described pin hole of adjacent two row, is called J; Centre distance with adjacent two described pin holes of a line is defined as K; The centre distance of adjacent two described pin holes of same row is defined as L; The arrangement of described pin hole makes the big or small r of I, J, K and L and described pin hole have following equation relation:
0＜I≤2r
L＝n×I，
K
2＝n
2×L
2-I
2
J
2＝L
2-I
2
N is the coefficient of described pin hole spacing for a change, 1 < n≤20.
6. a kind of confocal optical scanner according to claim 1 and 2, is characterized in that: described first reflecting surface of described scanning galvanometer is that plane reflection or concave reflection, described the second reflecting surface are plane reflection or concave reflection.
7. a kind of confocal optical scanner according to claim 6, is characterized in that: described the first reflecting surface and described the second reflecting surface are respectively on two scanning galvanometers.
8. a kind of confocal optical scanner according to claim 1 and 2, also comprises a slice dichroic beam splitter, between described light source and described pinhole array, sees through the light of described light source transmitting, reflects the fluorescence of described sample transmitting.
9. a kind of confocal optical scanner according to claim 1 and 2, also comprise a cut-off diaphragm, be positioned at the picture plane of described micro objective to described sample imaging, its role is to: in the time that described scanning galvanometer rotates to maximum angular position, described light source is launched and the light that sees through described pinhole array is stopped by described cut-off diaphragm, can not enter sample described in described microscope illumination.
10. a kind of confocal optical scanner according to claim 1 and 2, also comprise the controller that has signal and communication between and described battle array detecting device, rotated by described controller gated sweep galvanometer, make the time shutter of described battle array detecting device equal the integral multiple of the rotational time of described scanning galvanometer, and to make the startup of described scanning galvanometer rotation and the startup of described battle array detector exposure be synchronization.
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| CN104010122A (en) * | 2014-06-13 | 2014-08-27 | 广东欧珀移动通信有限公司 | Light sensing chip, camera and electronic product |
| CN104111243A (en) * | 2014-07-11 | 2014-10-22 | 江苏大学 | Fluorescence ratio measuring system and method |
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| CN108051368A (en) * | 2017-11-28 | 2018-05-18 | 华中科技大学 | A kind of periodicity sub-wavelength array of orifices sample template and its imaging method |
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