CN102818795B - Biological fluorescence microscopic detection instrument - Google Patents
Biological fluorescence microscopic detection instrument Download PDFInfo
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- CN102818795B CN102818795B CN201210255748.7A CN201210255748A CN102818795B CN 102818795 B CN102818795 B CN 102818795B CN 201210255748 A CN201210255748 A CN 201210255748A CN 102818795 B CN102818795 B CN 102818795B
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Abstract
Biological fluorescence microscopic detection instrument provided by the invention comprises exciting light lighting unit, exciting light and fluorescence isolated location, laser scan unit, micro-imaging unit, fluorescence detecting unit and control module.Between first beam expanding lens of exciting light lighting unit and the second beam expanding lens and the focus place being positioned at the first beam expanding lens is provided with illumination pin hole, between the imaging lens of fluorescence detecting unit and photomultiplier and the focus place being positioned at imaging lens is provided with imaging detection pin hole, effectively improve the lateral resolution of this instrument; Meanwhile, annular beam shaping component is set in the position of the microcobjective entrance pupil of micro-imaging unit, improves the axial resolution of this biological fluorescence microscopic detection instrument.
Description
Technical field
The present invention relates to microscopic detection instrument design and manufacture field, especially relate to a kind of biological fluorescence microscopic detection instrument.
Background technology
Total internal reflection micro-imaging technique and confocal microscopic image technology respectively have relative merits: total internal reflection micro-imaging has very high axial resolution, but its lateral resolution is lower; Confocal microscopic image has very high lateral resolution, but its axial resolution is poor.How to utilize in a microscope simultaneously axial resolution that total internal reflection micro-imaging is high and the high lateral resolution of confocal microscopic image particularly important.
Propose by total internal reflection fluorescent image and confocal images simply and the image processing apparatus correctly overlapped, program and microscope in application for a patent for invention CN201080024155.9, two cover microscopes are contained in this invention, a set of is total internal reflectance microscope, another set of is Laser Scanning Confocal Microscope, but two cover microscopes are separately work, the method switched by light path first obtains a set of microscopical image, reentry another set of microscopical image, two cover images overlap by the reference points in last computing machine recycling two cover images.Although the method generates the coincidence pattern picture of total internal reflection fluorescent image and confocal images, because two kinds of microscopes are successively work, three-dimension high-resolution image can not be obtained physically simultaneously; This invention successively will operate two microscope photographing images in addition, and will overlap to image, microscopic structure more complicated, the formation also more complicated of control system.
Summary of the invention
The object of the invention is: the biological fluorescence microscopic detection instrument providing a kind of integrated total internal reflection micro-imaging technique and confocal microscopic image technology, this biological fluorescence microscopic detection instrument is in transverse direction and axially have very high resolution.
Technical scheme of the present invention is: biological fluorescence microscopic detection instrument comprises exciting light lighting unit, exciting light and fluorescence isolated location, laser scan unit, micro-imaging unit, fluorescence detecting unit and control module; Described exciting light lighting unit comprises LASER Light Source, the first beam expanding lens and the second beam expanding lens; Be provided with illumination pin hole between described first beam expanding lens and the second beam expanding lens, and described illumination pin hole is positioned at the focus place of described first beam expanding lens; Described exciting light and fluorescence isolated location comprise exciting light color filter, dichroscope and fluorescence color filter; Described laser scan unit comprises and can make the X scanning galvanometer assembly of round rotary motion around X-axis and can make the Y scanning galvanometer assembly of round rotary motion around Y-axis; Described micro-imaging unit comprises scanning lens, cylinder mirror, annular beam shaping component and microcobjective, described annular beam shaping component comprises the annular optical filter being located at described microcobjective entrance pupil position, described annular optical filter has the ring belt area of endocyclic area by laser cutoff and transmission laser, and the equal transmissive fluorescence of described endocyclic area and ring belt area; The radius of described endocyclic area be not less than can just experiences total internal reflection time described microcobjective entrance pupil position critical radius; The entrance pupil position of described microcobjective and the reflecting surface of described X scanning galvanometer assembly and the reflecting surface of Y scanning galvanometer assembly are along the centre position phase conjugate of optical axis; Described fluorescence detecting unit comprises imaging lens and photomultiplier, is provided with imaging detection pin hole between described imaging lens and described photomultiplier, and described imaging detection pin hole is positioned at the focus place of described imaging lens; Object to be seen and described illumination pin hole and imaging detection pin hole are on conjugate position; The detectable fluorescence of described photomultiplier, and convert described fluorescence to electric signal;
Described first beam expanding lens, illumination pin hole, the second beam expanding lens, exciting light color filter, dichroscope, laser scan unit, scanning lens, cylinder mirror, annular optical filter and microcobjective are arranged along the optical axis starting from described LASER Light Source successively; And described microcobjective, annular optical filter, cylinder mirror, scanning lens, laser scan unit, dichroscope, fluorescence color filter, imaging lens, imaging detection pin hole and photomultiplier are arranged along starting from the fluorescence optical axis excited by object to be seen successively;
Described control module, is all electrically connected with described laser scan unit and described photomultiplier, for electric signal described in synchronous acquisition and described laser scan unit position coordinates and associate, to generate subject area image to be seen.
Below technique scheme is explained further:
The width-adjustable joint of described ring belt area.
Described microcobjective is the immersion oil object lens of infinite distance anaberration type large-numerical aperture.
Described control module is also electrically connected with described LASER Light Source, for controlling wavelength and the power of laser.
Advantage of the present invention is:
1. biological fluorescence microscopic detection instrument provided by the invention is between the first beam expanding lens and the second beam expanding lens and the focus place being positioned at the first beam expanding lens is provided with illumination pin hole, between imaging lens and photomultiplier and the focus place being positioned at imaging lens is provided with imaging detection pin hole, effectively improve the lateral resolution of this instrument; Meanwhile, annular beam shaping component is set in the position being positioned at microcobjective entrance pupil, improves the resolution of this biological fluorescence microscopic detection instrument in axis.
2. biological fluorescence microscopic detection instrument provided by the invention is about in the dotted region of Aili spot size because incident laser beam focuses on region area through micro-imaging unit, namely the fluorescent material in a very little very thin thickness region of area is only had to be excited, and the fluorescent material in other region can not be excited, namely eliminate the source of parasitic light from source, thus there is very high imaging signal to noise ratio (S/N ratio).
Accompanying drawing explanation
The biological fluorescence microscopic detection instrument structural representation that Fig. 1 provides for the embodiment of the present invention.
The structural representation of the annular optical filter that Fig. 2 provides for the embodiment of the present invention.
The annular beam that Fig. 3 provides for the embodiment of the present invention is by the paths schematic diagram of microcobjective.
The structural representation of the laser scan unit that Fig. 4 provides for the embodiment of the present invention.
Wherein: exciting light lighting unit 110, LASER Light Source 111, first beam expanding lens 112, second beam expanding lens 113, illumination pin hole 114, exciting light and fluorescence isolated location 120, exciting light color filter 121, dichroscope 122, fluorescence color filter 123, laser scan unit 130, X scanning galvanometer assembly 131, Y scanning galvanometer assembly 132, micro-imaging unit 140, scanning lens 141, cylinder mirror 142, annular beam shaping component 143, microcobjective 144, annular optical filter 1432, fluorescence detecting unit 150, imaging lens 151, photomultiplier 152, imaging detection pin hole 153, control module 160.
Embodiment
Please refer to Fig. 1 to Fig. 4.The light path indicating unidirectional arrow in Fig. 1 is laser propagation light path; The light path indicating four-headed arrow is expressed as fluorescence and propagates light path.
Embodiment: biological fluorescence microscopic detection instrument 100 comprises exciting light lighting unit 110, exciting light and fluorescence isolated location 120, laser scan unit 130, micro-imaging unit 140, fluorescence detecting unit 150 and control module 160.
Exciting light lighting unit 110 comprises LASER Light Source 111, first beam expanding lens 112, second beam expanding lens 113.Between the first beam expanding lens 112 and the second beam expanding lens 113 and the focus place being positioned at the first beam expanding lens 112 is provided with illumination pin hole 114.
Exciting light and fluorescence isolated location 120 comprise exciting light color filter 121, dichroscope 122, fluorescence color filter 123.Exciting light colour filter 121, for receiving laser, departs from the light beam of centre wavelength in filtering laser, and the light beam of central wavelength in transmission laser.Dichroscope 122 receives and reflects the laser through exciting light color filter 121 transmission, and transmission fluorescence.Fluorescence color filter 123 receives also transmission through the fluorescence of dichroscope 122 transmission, and ends laser.
Laser scan unit 130 comprises X scanning galvanometer assembly 131, Y scanning galvanometer assembly 132.X scanning galvanometer assembly 131 can do round rotary motion around X-axis.Y scanning galvanometer assembly 132 can do round rotary motion around Y-axis, and along with the rotary motion of X scanning galvanometer assembly 131, Y scanning galvanometer assembly 132, the angle after incoming laser beam reflection also will change thereupon.
Micro-imaging unit 140 comprises scanning lens 141, cylinder mirror 142, annular beam shaping component 143, microcobjective 144.Annular beam shaping component 143 comprises annular optical filter 1432.This annular optical filter 1432 is arranged at the entrance pupil position of microcobjective 144, has the ring belt area B of endocyclic area A by laser cutoff and transmission laser.The equal transmissive fluorescence of endocyclic area A and ring belt area B.The reflecting surface of the entrance pupil position of microcobjective 144 and the reflecting surface of X scanning galvanometer assembly 131 and Y scanning galvanometer assembly 132 is along the centre position phase conjugate of optical axis, and when namely X scanning galvanometer assembly and Y scanning galvanometer assembly are placed in zero field angle position, Fig. 4 crosses position and the microcobjective 144 entrance pupil position conjugate of M point vertical optical axis.
Fluorescence detecting unit 150 comprises imaging lens 151, photomultiplier 152.Between imaging lens 151 and photomultiplier 152 and the focus place being positioned at imaging lens 151 is provided with imaging detection pin hole 153.Object to be seen is on conjugate position with illumination pin hole 114 and imaging detection pin hole 153.The detectable fluorescence of photomultiplier 152, and convert fluorescence to electric signal.
Wherein, the first beam expanding lens 112, illumination pin hole 114, second beam expanding lens 113, exciting light color filter 121, dichroscope 122, laser scan unit 130, scanning lens 141, cylinder mirror 142, annular optical filter 1432, microcobjective 144 are arranged along the optical axis starting from LASER Light Source 111 successively; Microcobjective 144, annular optical filter 1432, cylinder mirror 142, scanning lens 141, laser scan unit 130, dichroscope 122, fluorescence color filter 123, imaging lens 151, imaging detection pin hole 153, photomultiplier 152 are arranged along starting from the fluorescence optical axis that object to be seen excites successively.
The collimation laser that LASER Light Source 111 is launched forms parallel laser beam through the first beam expanding lens 112, illumination pin hole 114, second beam expanding lens 113 after expanding successively; This collimated laser beam is shaped as annular beam through annular beam shaping component 143 successively after exciting light color filter 121, dichroscope 122, laser scan unit 130, scanning lens 141, cylinder mirror 142, this annular beam is gathered in object place to be seen through microcobjective 144, and excites object to be seen to produce fluorescence; This fluorescent light beam focuses on imaging detection pin hole 153 place successively after microcobjective 144, annular optical filter 1432, cylinder mirror 142, scanning lens 141, laser scan unit 130, dichroscope 122, fluorescence color filter 123, imaging lens 151, and photomultiplier 152 detects this fluorescence beam and converts thereof into electric signal.
Control module 160 and laser scan unit 130, photomultiplier 152 are electrically connected, the ultra-weak electronic signal that photomultiplier 152 exports amplifies by control module 160, and real-time sampling is carried out to the electric signal after amplifying, control X scanning galvanometer assembly 131, Y scanning galvanometer assembly 132 come and go rotate along X, Y-axis simultaneously, and the laser focusing point formed through microcobjective 144 can be moved in X, Y-direction.Control module 160 by collecting electric signal and laser scan unit 130X, the position coordinates of Y-direction associates, and generates the image of fluorescent material in a region.Control module 160 is also electrically connected at LASER Light Source 111, for controlling laser wavelength of incidence and power.
In this biological fluorescence microscopic detection instrument 100, microcobjective 144 is the immersion oil object lens of infinite distance anaberration type large-numerical aperture, the inner ring radius radius of a-quadrant (in the Fig. 2) due to annular beam be not less than microcobjective 144 can just experiences total internal reflection time entrance pupil position light beam critical radius, and cover glass 200 is roughly the same with the refractive index of oil 300, the annular beam entering microcobjective 144 is like this totally reflected at the intersection of the tissue solution 400 at cover glass 200 and object place to be seen, can not propagate through cover glass 200, but evanescent field (Tu3Zhong C district) can be formed at the intersection of cover glass 200 and tissue solution 400, evanescent wave can excite the fluorescence molecule of near interface, produce fluorescence.The width of adjustment annular optical filter 1432 ring belt area B can change the distributed depth of the evanescent field passing cover glass 200, thus can change the shooting depth of fluorescent material in Z-direction, can realize the adjustment of Z-direction different resolution.The frequency of evanescent wave is identical with incident light frequency, and its intensity (energy of unit area and unit interval) exponentially decays with the vertical range leaving interface:
I(z)＝I(0)e
-z/d
Can find out, the degree of depth z that the amplitude of transmitted electromagnetic field enters sample reduces quickly, and this electromagnetic field is only present near interface skim.D is theoretical length of penetration, and equal the distance to evanescent wave strength retrogression to interface numerical value 1/e from interface, d can be expressed as:
d＝(λ
0/4π)(n
1 2sin
2θ-n
2 2)
-1/2
D and incident angle (θ), wavelength X
0and tissue solution 400 refractive index (n
2) and the refractive index (n of cover glass 200
1) relevant.D increases with incident angle and reduces, and size and lambda1-wavelength are the same order of magnitude or less.Due to the unique property of evanescent field, make the region of fluorescence excitation very near interphase (about 100nm).The fluorescence apart from interphase more far region can not be excited like this, thus the minimum fluorescence imaging of ground unrest can be realized, make biological fluorescence microscopic detection instrument 100 axially have very high resolution.
In this biological fluorescence microscopic detection instrument 100, by the conbined usage of throw light on pin hole 114 and imaging detection pin hole 153, realize point-to-point illumination and point-to-point imaging.When not considering noise, optically conventional point spread function descriptive system imaging resolution, for laser-scanning confocal system, the final point spread function of system is described by following formula:
PSF
tot(x，y，z)＝PSF
ill(x，y，z)·PSF
det(x，y，z)
Wherein PSF
illcorresponding laser illuminator point at the point spread function of object space, PSF
detthe point spread function of corresponding imaging detection light path.Due to the effect of the pin hole 114 that throws light on, incident laser beam is by forming a very little point-like field of illumination (side's of illumination point spread function) at cover glass 200 and object intersection to be seen after each unit, the use of imaging detection pin hole 153 is to the further shaping of imaging detection side's point spread function, the imaging point spread function of whole biological fluorescence microscopic detection instrument 100 is made up of the product of the side's of illumination point spread function and detection side's point spread function, because the imaging point spread function intensity distribution range after product narrows, thus system has very high lateral resolution.
Be about in the dotted region of Aili spot size because incident laser beam focuses on region area through micro-imaging unit 140 in this biological fluorescence microscopic detection instrument 100, namely the fluorescent material in a very little very thin thickness region of area is only had to be excited, and the fluorescent material in other region can not be excited, namely eliminate the source of parasitic light from source, thus system has very high imaging signal to noise ratio (S/N ratio).
Certain biological fluorescence microscopic detection instrument of the present invention also can have multiple conversion and remodeling, is not limited to the concrete structure of above-mentioned embodiment.In a word, protection scope of the present invention should comprise those apparent conversion or alternative and remodeling to those skilled in the art.
Claims (2)
1. a biological fluorescence microscopic detection instrument, is characterized in that, comprises exciting light lighting unit, exciting light and fluorescence isolated location, laser scan unit, micro-imaging unit, fluorescence detecting unit, control module and cover glass;
Described exciting light lighting unit comprises LASER Light Source, the first beam expanding lens, the second beam expanding lens; Be provided with illumination pin hole between first beam expanding lens, the second beam expanding lens, and described illumination pin hole is positioned at the focus place of described first beam expanding lens; Described exciting light and fluorescence isolated location comprise exciting light color filter, dichroscope, fluorescence color filter; Described laser scan unit comprises and can make the X scanning galvanometer assembly of round rotary motion, can make the Y scanning galvanometer assembly of round rotary motion around Y-axis around X-axis; Described micro-imaging unit comprises scanning lens, cylinder mirror, annular beam shaping component, microcobjective, described annular beam shaping component comprises the annular optical filter of the entrance pupil position being located at described microcobjective, described annular optical filter has the ring belt area of endocyclic area by laser cutoff and transmission laser, the equal transmissive fluorescence of this endocyclic area and ring belt area; The radius of described endocyclic area be not less than can just experiences total internal reflection time described microcobjective entrance pupil position critical radius; The width-adjustable joint of described ring belt area; Described microcobjective is the immersion oil object lens of infinite distance anaberration type large-numerical aperture; Described cover glass is identical with the refractive index of the oil of described immersion oil object lens, the annular beam entering described microcobjective is totally reflected at the intersection of the tissue solution at described cover glass and object place to be seen, described annular beam forms evanescent field at the intersection of described cover glass and described tissue solution, and the width adjusting described ring belt area can change the distributed depth of described evanescent field; The entrance pupil position of described microcobjective and the reflecting surface of described X scanning galvanometer assembly and the reflecting surface of Y scanning galvanometer assembly are along the centre position phase conjugate of optical axis; Described fluorescence detecting unit comprises imaging lens, photomultiplier, is provided with imaging detection pin hole between this imaging lens and described photomultiplier, and described imaging detection pin hole is positioned at the focus place of described imaging lens; Object to be seen and described illumination pin hole and imaging detection pin hole are on conjugate position; The detectable fluorescence of described photomultiplier, and convert described fluorescence to electric signal;
Described first beam expanding lens, illumination pin hole, the second beam expanding lens, exciting light color filter, dichroscope, laser scan unit, scanning lens, cylinder mirror, annular optical filter and microcobjective are arranged along the optical axis starting from described LASER Light Source successively, and described microcobjective, annular optical filter, cylinder mirror, scanning lens, laser scan unit, dichroscope, fluorescence color filter, imaging lens, imaging detection pin hole and photomultiplier are arranged along starting from the fluorescence optical axis excited by object to be seen successively;
Described control module and laser scan unit, photomultiplier are all electrically connected, for electric signal described in synchronous acquisition and described laser scan unit position coordinates and associate, to generate subject area image to be seen.
2. biological fluorescence microscopic detection instrument according to claim 1, is characterized in that, described control module is also electrically connected with described LASER Light Source, for controlling wavelength and the power of laser.
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| US9746412B2 (en) | 2012-05-30 | 2017-08-29 | Iris International, Inc. | Flow cytometer |
| CN104391370B (en) * | 2014-11-04 | 2017-09-12 | 温州生物材料与工程研究所 | The imaging converting system of flat field scanning lens working face and surgical operation microscope working face |
| CN105675553B (en) * | 2015-12-14 | 2018-09-25 | 中国人民解放军军事医学科学院卫生装备研究所 | Trace microbial rapid detection system |
| CN105527261B (en) * | 2015-12-30 | 2018-07-17 | 中国科学院苏州生物医学工程技术研究所 | A kind of microscopical multi-modal scanning means of two-photon fluorescence |
| CN107037016A (en) * | 2016-02-04 | 2017-08-11 | 北京世纪桑尼科技有限公司 | A kind of confocal optical scanner |
| CN107014793B (en) * | 2017-04-21 | 2019-07-30 | 浙江大学 | One kind is based on double galvanometer doublet multi-mode wide fields super-resolution micro imaging system |
| CN107515209A (en) * | 2017-10-02 | 2017-12-26 | 西南石油大学 | A kind of Multifunction fluorescent sample lights testboard |
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