CN102841083A - Method and system of laser scanning phase-microscope imaging - Google Patents
Method and system of laser scanning phase-microscope imaging Download PDFInfo
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Abstract
本发明涉及一种激光扫描位相显微成像方法及系统，它包括有荧光显微镜、激光共聚焦扫描系统和光电探测系统；将激光共聚焦扫描系统放置在荧光显微镜入光孔，将光电探测系统放置在激光共聚焦扫描系统的出光方向；调节各光学元件之间的角度；将样品切片放置在试验台上；在载玻片的顶部添加荧光介质；激光器发出激发光经二向色镜、X方向扫描振镜、Y方向扫描振镜、扫描镜和管镜聚焦扩束后发射到物镜，并聚焦在生物样品上；聚焦在生物样品的激发光透过生物样品照射在荧光介质上，荧光介质被激发光激发发射荧光；荧光照射并透过生物样品沿着原光路返回，出射的荧光发射到滤波片并经会聚透镜聚焦进入针孔，经针孔出射的荧光由光电探测系统接收处理得到生物样品的浮雕状结构。本发明可以广泛应用于生物样品成像过程中。
The invention relates to a laser scanning phase microscopic imaging method and system, which includes a fluorescence microscope, a laser confocal scanning system and a photoelectric detection system; the laser confocal scanning system is placed in the light entrance of the fluorescence microscope, and the photoelectric detection system is placed In the light output direction of the laser confocal scanning system; adjust the angle between each optical element; place the sample slice on the test bench; add a fluorescent medium on the top of the glass slide; the laser emits excitation light through the dichroic mirror, X direction The scanning galvanometer, the Y-direction scanning galvanometer, the scanning mirror and the tube mirror focus and expand the beam and emit it to the objective lens and focus on the biological sample; the excitation light focused on the biological sample is irradiated on the fluorescent medium through the biological sample, and the fluorescent medium is The excitation light excites and emits fluorescence; the fluorescence is irradiated and returns along the original optical path through the biological sample, the outgoing fluorescence is emitted to the filter and focused into the pinhole through the converging lens, and the fluorescence emitted through the pinhole is received and processed by the photoelectric detection system to obtain the biological sample relief-like structure. The invention can be widely used in the imaging process of biological samples.
Description
Technical field
The present invention relates to a kind of optical imaging method and system, particularly about a kind of laser scanning position phase micro imaging method and system that is applicable to the transparent organism imaging of tissue.
Background technology
The laser scanning co-focusing microtechnic is to utilize the pin hole that places detector preceding; Effectively the non-focal plane light of shielding is to the influence of sample image quality; Realize the imaging process that spot scan, point are surveyed; But because it does not have the ability that the sample phase information is changed into strength information, therefore can not carry out the structure imaging of non-fluorescent label, need merge and to realize point by point scanning/detection method to the transparent organism sample with other micro imaging method to the transparent organism sample.
Biological tissue's micro imaging method mainly comprises in the prior art: 1, differential interference differs micro imaging method and utilizes two wollaston prisms (Wollaston Prism); The polarizer (Polarizer) and analyzer (Analyzer); Based on the wide field polarized light transparent organism tissue is carried out to picture; Imaging results can present the embossment shape structure of biological tissue; But above-mentioned micro imaging method need produce polarized light by extra interpolation polarizer in light path, and utilizes the angle of shear of two bundle polarized lights formation to carry out the detection of phasic difference branch, and employed element costs an arm and a leg and can not survey the sample with birefringent characteristic.Simultaneously, the wide field imaging needs to use the CCD camera to carry out data acquisition, therefore is unfavorable for merging with the laser scanning co-focusing microtechnic that adopts point probe.2, the oblique illumination micro imaging method is to utilize Huffman optical modulator (Hoffman Modulator) and slit diaphragm (Slit Stop); Light source based on the wide field oblique incidence is carried out to picture to the transparent organism tissue; Imaging results can present the embossment shape structure of biological tissue; But owing to need to insert optical modulator and slit diaphragm in the light path of above-mentioned micro imaging method; Therefore in experimentation, be unfavorable for the switching between different modalities, simultaneously also because the wide field imaging needs to use the CCD camera to carry out data acquisition, so be unfavorable for merging with the laser scanning co-focusing microtechnic that adopts point probe.
Summary of the invention
To the problems referred to above; The purpose of this invention is to provide a kind of unmarked imaging that can realize to the transparent organism sample; Not only can make things convenient between the different modalities and switch, and image contrast and signal to noise ratio (S/N ratio) are all than higher laser scanning position micro imaging method and system mutually.
For realizing above-mentioned purpose; The present invention takes following technical scheme: a kind of laser scanning position phase micro imaging method may further comprise the steps: 1) be provided with one include fluorescent microscope, laser confocal scanning system and Photodetection system laser scanning position micro imaging system mutually; Wherein, said fluorescent microscope includes object lens, Guan Jing and scanning mirror; Said laser confocal scanning system includes laser instrument, dichroic mirror, directions X scanning galvanometer, Y scanning direction galvanometer, filter plate, convergent lens and pin hole; 2) the laser confocal scanning system is placed on the incidence hole place of fluorescent microscope, Photodetection system is placed on the light direction of laser confocal scanning system, make the height of height and fluorescent microscope incidence hole axis of laser emitting exciting light suitable; 3) regulate angle between each optical element, make imaging system satisfy the image conjugate relation of optical microscope system; 4) sample section is placed on the testing table of fluorescent microscope, said sample section comprises cover glass, biological sample and microslide; 5) on microslide, add fluorescent media; 6) the laser instrument launching excitation light carries out beam split through dichroic mirror; The exciting light of outgoing becomes the grating face through the directions X scanning galvanometer with Y scanning direction vibration mirror scanning successively; And it is transmitted into object lens after scanning mirror and pipe mirror focus on to expand bundle, and focus on the biological sample through cover glass; 7) exciting light that focuses on biological sample sees through biological sample irradiation fluorescent media, the fluorescent media optical excitation emitting fluorescence that is excited; 8) fluorescence shines and sees through biological sample and returns along original optical path; Promptly be transmitted into dichroic mirror and carry out beam split through object lens, Guan Jing, scanning mirror, Y scanning direction galvanometer, directions X scanning galvanometer successively; The fluorescent emission of outgoing focuses on to filter plate and through convergent lens and gets into pin hole, handles the embossment shape structure that obtains biological sample through the fluorescence of pin hole outgoing by the Photodetection system reception.
The angle concrete operations of regulating between each optical element in the said step 3) are: regulate the angle between directions X scanning galvanometer and the object lens, making and getting into object lens preceding excitation light direction of principal axis and the angle between the object lens axis is θ, α
1<θ<α
2, wherein, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
The angle concrete operations of regulating between each optical element in the said step 3) are: regulate the angle between directions X scanning galvanometer and the object lens; The preceding excitation light direction of principal axis of entering object lens is overlapped with the axis of object lens; And the position between adjusting convergent lens and the pin hole; Making the fluorescence and the angle between the pin hole axis that focus on through convergent lens is β, α
1γ<β<α
2γ, wherein, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
The angle concrete operations of regulating between each optical element in the said step 3) are: regulate the angle between directions X scanning galvanometer and the object lens, making the angle between the axis that gets into preceding excitation light direction of principal axis of object lens and object lens is δ
1, regulate the position between convergent lens and the pin hole simultaneously, making the fluorescence and the angle between the pin hole axis that focus on through convergent lens is δ
2, wherein, δ
10, δ
20, α
1<δ
1+ δ
2/ γ<α
2, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
Realize a kind of laser scanning position phase micro imaging system of said method, it is characterized in that: it comprises fluorescent microscope, laser confocal scanning system and Photodetection system, and said fluorescent microscope comprises object lens, Guan Jing and scanning mirror; Said laser confocal scanning system comprises laser instrument, dichroic mirror, directions X scanning galvanometer, Y scanning direction galvanometer, filter plate, convergent lens and pin hole; Said laser confocal scanning system is arranged on the incidence hole place of said fluorescent microscope; Said Photodetection system is arranged on the light direction of said pin hole; Said laser instrument launching excitation light carries out beam split to said dichroic mirror; Exciting light through said dichroic mirror outgoing becomes grating face along directions X with the Y scanning direction with Y scanning direction galvanometer through said directions X scanning galvanometer successively; The grating face focuses on through said scanning mirror and gets into said fluorescent microscope, and successively through said Guan Jing and object lens focusing in biological sample and shine fluorescent media, through the fluorescence irradiation of excitation and through biological sample; Penetrate along said object lens, Guan Jing and scanning mirror; And be transmitted into said dichroic mirror through said Y scanning direction galvanometer and directions X scanning mirror successively and carry out beam split, focus on through said filter plate and convergent lens through the fluorescence of said dichroic mirror outgoing and get into said pin hole, and shine the fluorescence imaging of accomplishing biological sample on the said Photodetection system through said pin hole.
Getting into preceding excitation light direction of principal axis of said object lens and the angle between the said object lens axis is θ, and α
1<θ<α
2, wherein, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
The excitation light direction of principal axis that gets into before the said object lens overlaps with said object lens axis, and is β through the fluorescence optical axis of said convergent lens focusing and the angle between the said pin hole axis, α
1γ<β<α
2γ, wherein, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
The angle that gets between the axis of excitation light direction of principal axis and said object lens before the said object lens is δ
1, and the fluorescence that focuses on through said convergent lens and the angle between the said pin hole axis are δ
2, wherein, δ
10, δ
20, α
1<δ
1+ δ
2/ γ<α
2, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
The present invention is owing to take above technical scheme; It has the following advantages: 1, the present invention is owing to be provided with fluorescent microscope, laser confocal scanning system and Photodetection system; And (sample rear) is provided with fluorescent media on microslide; When biological sample to be measured is carried out laser scanning position phase micro-imaging; Excitation fluorescence irradiating biological sample also passes through to regulate the angle between each optical element, can accomplish the embossment shape imaging of transparent organism sample structure, has realized the unmarked imaging to the transparent organism sample.2, the present invention is owing to utilize the fluorescent media excited fluorescent irradiating biological sample that is arranged on the sample rear; Realize the structure embossment shape imaging of transparent sample; And also can utilize the fluorescence of the mark of biological sample own, realize the conventional fluorescent imaging, therefore can in the scanning optical imaging, accomplish biological sample structure imaging or fluorescence imaging; Handoff procedure is simple, has greatly made things convenient between the different modalities and has switched.3, the present invention is owing to adopt laser confocal scanning/detection mode; Therefore receive only the sample message that is positioned on the object lens focal plane; Shielded of the influence of non-focal plane light effectively to image quality; Image contrast is high, and signal to noise ratio (S/N ratio) is high, makes point by point scanning/detection method can be applicable to the imaging of transparent organism sample.4, the present invention compared with prior art; Owing to do not need differential interference to differ distinctive optical element in micro-imaging technique and the oblique illumination micro-imaging technique; Therefore greatly reduce the complicacy of micro imaging system, the optical element of having avoided inserting in the light path makes experimental system and experimentation complicated.The present invention can be widely used in the imaging process of biological sample.
Description of drawings
Fig. 1 is a laser scanning of the present invention position phase micro imaging system structural representation;
Fig. 2 is the angle amplification profile synoptic diagram of the present invention when laser oblique incidence;
Fig. 3 is a pin hole mitered time receiving angle amplification profile synoptic diagram of the present invention.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is carried out detailed description.
As shown in Figure 1, laser scanning of the present invention position phase micro imaging system comprises a fluorescent microscope 1, a laser confocal scanning system 2 and a Photodetection system 3; Fluorescent microscope 1 comprises object lens 11, pipe mirror 12 and scanning mirror 13; Laser confocal scanning system 2 comprises laser instrument 21, dichroic mirror 22, directions X scanning galvanometer 23, Y scanning direction galvanometer 24, filter plate 25, convergent lens 26 and pin hole 27; Laser confocal scanning system 2 is positioned at the incidence hole place of fluorescent microscope 1, and Photodetection system 3 is positioned at the light direction of pin hole 27, is used to receive the fluorescence through the biological sample transmission.Reflection or transmission (beam split) take place to dichroic mirror 22 in laser instrument 21 launching excitation lights; Excitation light emission through dichroic mirror 22 reflections is scanned into grid stroke (line source) to directions X scanning galvanometer 23 with exciting light; And it is transmitted into Y scanning direction galvanometer 24 grid stroke is scanned into grating face (area source); The grating face focuses on through scanning mirror 13 and gets into fluorescent microscope 1; And focus on biological sample through pipe mirror 12 with object lens 11 successively, penetrate fluorescent microscopes 1 through the fluorescence of excitation along object lens 11, pipe mirror 12 and scanning mirror 13, and be transmitted into dichroic mirror 22 through Y scanning direction galvanometer 24 and directions X scanning mirror 23 successively transmission or reflections take place; The fluorescence of transmission focuses on convergent lens 26 through filter plate 25 and gets into pin hole 27, and shines the fluorescence imaging of accomplishing biological sample on the Photodetection system 3 through pin hole 27.
In the foregoing description, Photodetection system 3 can select to have the optical element of photodetection function according to actual needs, does not limit at this.
Among above-mentioned each embodiment, filter plate 25 is selected based on the wavelength correspondence of the fluorescent media institute excited fluorescent that is adopted.
Among above-mentioned each embodiment, the effect of dichroscope 22 is beam split, can adopt reflection exciting light transmission fluorescence, also can adopt transmission exciting light reflected fluorescent light, in concrete experiment, can set based on actual needs.
As shown in Figure 1; When reality is carried out fluorescence imaging to biological sample; Fluorescent microscope 1 can adopt the mode of just putting fluorescent microscope or inverted fluorescence microscope to accomplish imaging; Both differences only are that light path is spun upside down, and the present invention is that specific embodiment detailed description laser scanning position phase micro imaging system carries out laser scanning position phase micro imaging method to biological sample to be measured with inversion type fluorescent microscope 1, may further comprise the steps:
1) laser confocal scanning system 2 is placed on the incidence hole place of fluorescent microscope 1; Photodetection system 3 is placed on the light direction of laser confocal scanning system 2; And the height of the shoot laser of assurance laser instrument 21 and the height of fluorescent microscope 1 incidence hole axis be (approximately equal) quite, makes exciting light get into 1 pair of biological sample of fluorescent microscope through incidence hole and shines.
2) according to actual needs, regulate the angle between each optical element, make imaging system satisfy the image conjugate relation of optical microscope system.
3) sample section 4 is placed on the testing table of fluorescent microscope 1, sample section 4 is followed successively by cover glass 41, biological sample 42 and microslide 43 from bottom to top.
4) add fluorescent media 5 at the top of microslide 43, this fluorescent media 5 is can be by the effective excited fluorescent material of incident exciting light.
5) laser instrument 21 sends exciting light and carries out beam split through dichroic mirror 22; Exciting light through dichroic mirror 22 outgoing becomes grating face along directions X with the Y scanning direction with Y scanning direction galvanometer 24 through directions X scanning galvanometer 23 successively; And it is focused on through scanning mirror 13 and pipe mirror 12 be transmitted into object lens 11 after expanding bundle; And focus on the biological sample 42 through cover glass 41, accomplish laser scanning to biological sample.
6) because biological sample itself is near transparence, the most of biological sample 42 that sees through of exciting light that therefore focuses on biological sample 42 is radiated on the fluorescent media 5, and the fluorescent media 5 optical excitation emitting fluorescence that is excited is for biological sample 42 provides illumination.
7) fluorescence irradiation and see through biological sample 42 and return along original optical path; Promptly be transmitted into dichroic mirror 22 and carry out beam split through object lens 11, pipe mirror 12, scanning mirror 13, Y scanning direction galvanometer 24, directions X scanning galvanometer 23 successively; Since the wavelength of fluorescence with excite light wavelength different; Therefore fluorescence shines filter plate 25 and focuses on entering pin hole 27 through convergent lens 26 through dichroic mirror 22, handles the embossment shape structure that forms biological sample by Photodetection system 3 receptions and through existing image processing method through the fluorescence of pin hole 27 outgoing.
As shown in Figure 2; In the foregoing description; Therefore for the imaging results that makes biological sample can present the embossment shape structure of biological tissue, can adopt fluorescence is that biological sample provides wide field oblique fire light source, step 2) in the angle concrete operations regulated between each optical element be: regulate the angle between directions X scanning galvanometer 23 and the object lens 11; Making the angle between the axis of the excitation light direction of principal axis that gets into before the object lens 11 and object lens 11 is θ, and α
1<θ<α
2, α
1Be exciting light scanning field of view angle (the exciting light deflection angle corresponding), α with the sample sweep limit
2Be the numerical aperture angle of object lens 11, wherein, sin α
2=NA/n, NA are the numerical aperture of object lens, and n is a medium refraction index.
As shown in Figure 3; In the foregoing description; Image conjugate relation according to optical imagery can be known; Step 2) the angle concrete operations of regulating between each optical element in are: regulate the angle between directions X scanning galvanometer 23 and the object lens 11, when entering object lens 11 preceding excitation light direction of principal axis are overlapped with the axis of object lens 11 (angle between the axis of the optical axis direction of exciting light and object lens 11 is 0), regulate the position between convergent lens 26 and the pin hole 27; Making the fluorescence optical axis and the angle between the pin hole axis that focus on through convergent lens 26 is β, and variation range is α
1γ<β<α
2γ is according to the magnification relation of optical system, angular magnification γ=f
1/ f
2, f
1Be the focal length of object lens 11, f
2Be the focal length of convergent lens 26, therefore, when laser normal incidence,, also can make the imaging results of biological sample present the embossment shape structure of biological tissue through being provided with through the fluorescence optical axis of convergent lens focusing 26 and the angle between pin hole 27 axis.
In the foregoing description; Step 2) regulate between each optical element the angle concrete operations also can for: regulate the angle between directions X scanning galvanometer 23 and the object lens 11, making the angle between the axis that gets into preceding excitation light direction of principal axis of object lens 11 and object lens 11 is δ
1(δ
10), regulate the position between convergent lens 26 and the pin hole 27 simultaneously, making through the fluorescence optical axis of convergent lens 26 focusing and the angle between pin hole 27 axis is δ
2(δ
20), when satisfying α
1<δ
1+ δ
2/ γ<α
2The time, also can be so that the imaging results of biological sample presents the embossment shape structure of biological tissue.
Among above-mentioned each embodiment, the diameter of pin hole 27 can adopt micron dimension, and purpose gets into Photodetection system 3 in order better to avoid from non-focal plane light.
Among above-mentioned each embodiment; All optical device of the present invention in use all can adopt corresponding outside support to position; The present invention does not limit the particular location of each optical element; Can adjust according to concrete requirement of experiment, but the light path that all optical element combination form propagation must be consistent with light path propagation of the present invention, satisfy irradiation and the detection requirement of the present invention biological sample.
Above-mentioned each embodiment only is used to explain the present invention; Wherein step of the position of each optical element, implementation method etc. all can change to some extent; Every equivalents of on the basis of technical scheme of the present invention, carrying out and improvement all should not got rid of outside protection scope of the present invention.
Claims (8)
1. laser scanning position phase micro imaging method may further comprise the steps:
1) be provided with one include fluorescent microscope, laser confocal scanning system and Photodetection system laser scanning position micro imaging system mutually; Wherein, said fluorescent microscope includes object lens, Guan Jing and scanning mirror; Said laser confocal scanning system includes laser instrument, dichroic mirror, directions X scanning galvanometer, Y scanning direction galvanometer, filter plate, convergent lens and pin hole;
2) the laser confocal scanning system is placed on the incidence hole place of fluorescent microscope, Photodetection system is placed on the light direction of laser confocal scanning system, make the height of height and fluorescent microscope incidence hole axis of laser emitting exciting light suitable;
3) regulate angle between each optical element, make imaging system satisfy the image conjugate relation of optical microscope system;
4) sample section is placed on the testing table of fluorescent microscope, said sample section comprises cover glass, biological sample and microslide;
5) on microslide, add fluorescent media;
6) the laser instrument launching excitation light carries out beam split through dichroic mirror; The exciting light of outgoing becomes the grating face through the directions X scanning galvanometer with Y scanning direction vibration mirror scanning successively; And it is transmitted into object lens after scanning mirror and pipe mirror focus on to expand bundle, and focus on the biological sample through cover glass;
7) exciting light that focuses on biological sample sees through biological sample irradiation fluorescent media, the fluorescent media optical excitation emitting fluorescence that is excited;
8) fluorescence shines and sees through biological sample and returns along original optical path; Promptly be transmitted into dichroic mirror and carry out beam split through object lens, Guan Jing, scanning mirror, Y scanning direction galvanometer, directions X scanning galvanometer successively; The fluorescent emission of outgoing focuses on to filter plate and through convergent lens and gets into pin hole, handles the embossment shape structure that obtains biological sample through the fluorescence of pin hole outgoing by the Photodetection system reception.
2. a kind of laser scanning as claimed in claim 1 position phase micro imaging method; It is characterized in that: the angle concrete operations of regulating between each optical element in the said step 3) are: regulate the angle between directions X scanning galvanometer and the object lens; Making and getting into object lens preceding excitation light direction of principal axis and the angle between the object lens axis is θ, α
1<θ<α
2, wherein, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
3. a kind of laser scanning as claimed in claim 1 position phase micro imaging method; It is characterized in that: the angle concrete operations of regulating between each optical element in the said step 3) are: regulate the angle between directions X scanning galvanometer and the object lens; The preceding excitation light direction of principal axis of entering object lens is overlapped with the axis of object lens; And regulate the position between convergent lens and the pin hole, making the fluorescence and the angle between the pin hole axis that focus on through convergent lens is β, α
1γ<β<α
2γ, wherein, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
4. a kind of laser scanning as claimed in claim 1 position phase micro imaging method; It is characterized in that: the angle concrete operations of regulating between each optical element in the said step 3) are: regulate the angle between directions X scanning galvanometer and the object lens, making the angle between the axis that gets into preceding excitation light direction of principal axis of object lens and object lens is δ
1, regulate the position between convergent lens and the pin hole simultaneously, making the fluorescence and the angle between the pin hole axis that focus on through convergent lens is δ
2, wherein, δ
10, δ
20, α
1<δ
1+ δ
2/ γ<α
2, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
5. realize a kind of laser scanning position phase micro imaging system like claim 1 or 2 or 3 or 4 said methods; It is characterized in that: it comprises fluorescent microscope, laser confocal scanning system and Photodetection system, and said fluorescent microscope comprises object lens, Guan Jing and scanning mirror; Said laser confocal scanning system comprises laser instrument, dichroic mirror, directions X scanning galvanometer, Y scanning direction galvanometer, filter plate, convergent lens and pin hole; Said laser confocal scanning system is arranged on the incidence hole place of said fluorescent microscope; Said Photodetection system is arranged on the light direction of said pin hole; Said laser instrument launching excitation light carries out beam split to said dichroic mirror; Exciting light through said dichroic mirror outgoing becomes grating face along directions X with the Y scanning direction with Y scanning direction galvanometer through said directions X scanning galvanometer successively; The grating face focuses on through said scanning mirror and gets into said fluorescent microscope, and successively through said Guan Jing and object lens focusing in biological sample and shine fluorescent media, through the fluorescence irradiation of excitation and through biological sample; Penetrate along said object lens, Guan Jing and scanning mirror; And be transmitted into said dichroic mirror through said Y scanning direction galvanometer and directions X scanning mirror successively and carry out beam split, focus on through said filter plate and convergent lens through the fluorescence of said dichroic mirror outgoing and get into said pin hole, and shine the fluorescence imaging of accomplishing biological sample on the said Photodetection system through said pin hole.
6. a kind of laser scanning as claimed in claim 5 position phase micro imaging system is characterized in that: getting into preceding excitation light direction of principal axis of said object lens and the angle between the said object lens axis is θ, and α
1<θ<α
2, wherein, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
7. a kind of laser scanning as claimed in claim 5 position phase micro imaging system; It is characterized in that: get into the preceding excitation light direction of principal axis of said object lens and overlap with said object lens axis; And through the fluorescence optical axis of said convergent lens focusing and the angle between the said pin hole axis is β, α
1γ<β<α
2γ, wherein, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
8. a kind of laser scanning as claimed in claim 5 position phase micro imaging system is characterized in that: the angle that gets between the axis of excitation light direction of principal axis and said object lens before the said object lens is δ
1, and the fluorescence that focuses on through said convergent lens and the angle between the said pin hole axis are δ
2, wherein, δ
10, δ
20, α
1<δ
1+ δ
2/ γ<α
2, γ is an angular magnification, α
1Be exciting light scanning field of view angle, α
2Numerical aperture angle for object lens.
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| CN103604787A (en) * | 2013-11-14 | 2014-02-26 | 北京大学 | Laser scanning phase microimaging method and system |
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