CN102818768A - Multifunctional biomedical microscope - Google Patents
Multifunctional biomedical microscope Download PDFInfo
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- CN102818768A CN102818768A CN2012102667006A CN201210266700A CN102818768A CN 102818768 A CN102818768 A CN 102818768A CN 2012102667006 A CN2012102667006 A CN 2012102667006A CN 201210266700 A CN201210266700 A CN 201210266700A CN 102818768 A CN102818768 A CN 102818768A
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Abstract
The invention relates to a multifunctional biomedical microscope with the functions of laser scanning confocal imaging, fluorescence imaging and optical coherence tomography. The multifunctional biomedical microscope is composed of a light source component, a two-dimensional scanning component, a zoom objective component, a confocal signal detecting component, an interference arm component and an interference signal detecting component. According to the multifunctional biomedical microscope, a confocal image of a sample is obtained by the introduction of a visible-light source lighting sample, a fluorescence image of the sample is obtained by the introduction of a fluorescence light source lighting sample, and an optical coherence tomography image of the sample is obtained by the introduction of a broadband low-coherence near-infrared laser light source lighting sample, and the multifunctional biomedical microscope is realized so as to obtain a micro-scale three-dimensional organization structure image of a transparent or non-transparent sample.
Description
Technical field
The present invention relates to a kind of multi-functional biomedical microscope, particularly a kind of biomedical tissue that is used for forms images, and can obtain the multi-functional biomedical microscope of the burnt image of copolymerization, fluoroscopic image and optical coherence tomography image.
Background technology
The notion of the burnt microtechnic of copolymerization (Confocal Microscopy) at first puts forward (Marvin Minsky. " Memo iron inventing confocal scanning microscope " by the Minsky of the U.S. in late 1950s; Scanning; 1988; 128-138), P.Davidovits subsequently, multidigit scholars such as A.F.Slomba and C.J.R.Sheppard have carried out finer research to confocal imaging.Laser confocal microscope system; Particularly the confocal fluorescent microscopic system of combined with fluorescent probe Detection Techniques because the employing system that makes of detecting pinhole and shorter wavelength laser has higher lateral resolution (about 100nm) and longitudinal frame (about 50nm), is widely used in the detection of biomedical tissue; Can obtain its inner structural images; Can also observe active somatic cell, obtain the information in the living cells, and the information that obtains is carried out quantitative test.But on the one hand, detecting pinhole has also limited the detection of flashlight when getting rid of parasitic light, and on the other hand, the through characteristic of short wavelength laser causes vertical measurement range of Laser Scanning Confocal Microscope on the low side, only 200-500um not as the long wavelength.
David Huang equals proposition optical coherence tomography (Optical Coherence Tomography in 1991; Be called for short OCT), and adopt this technology successfully to human eye retina's microstructure and coronary imaging (D.Huang, E.A.Swanson; Et al. " Optical coherence tomography "; Science, 1991,254:1178-1181).Thereafter David Huang etc. in the development of OCT technology, done more research (Huang D et al.Laser Surg Med 1991,11:5).Optical coherence tomography has improved the signal noise ratio level of system greatly owing to adopted the interference detection technology, and its investigation depth can reach 2-3mm, than the big one magnitude of Laser Scanning Confocal Microscope.But the finite bandwidth of low-coherence light source has limited its longitudinal frame, and the employing of long wavelength laser has simultaneously also reduced the lateral resolution of system.
Can know that in sum the detectable degree of depth of existing laser scanning confocal microscopy is less, and there is the not high shortcoming of resolution in Optical Coherence Tomography Imaging Technology, the two all demands urgently improving.
Contrast international and domestic technological achievement in burnt micro-imaging of copolymerization and optical coherent chromatographic imaging field; The present invention on this basis; A kind of new laser confocal microscope system is proposed and based on the multi-functional biomedical microscopie unit of optical coherent chromatographic imaging; Introduce a plurality of light source assembly illumination testing samples, realize laser co-focusing imaging, fluorescence imaging and optical coherent chromatographic imaging testing sample.
Summary of the invention
Technology of the present invention is dealt with problems:
1) overcomes the more shallow shortcoming of the single laser confocal microscope chromatography degree of depth; 2) overcome the horizontal and lower shortcoming of axial resolution of single optical coherence tomography system.
Technical solution of the present invention:
A kind of multi-functional biomedical microscope is made up of light source assembly, two-dimensional scan assembly, change times objective lens unit, confocal acquisition of signal assembly, interference arm component and interference signal probe assembly.The visible light that utilizes light source assembly is as lighting source; Treat the observing samples surface through a two-dimensional scan assembly and a change times objective lens unit back lighting; The flashlight that reflects from sample surfaces is received by confocal acquisition of signal assembly; Obtain the face scan image of sample, the layer of each degree of depth through can obtaining sample to moving axially of sample is cut image, and the layer of each degree of depth is cut image obtains sample after rebuilding 3-D view; Treat observing samples from the light signal illumination that the fluorescence light source of light source assembly sends, the layer that can obtain each degree of depth of sample is cut image and three-dimensional fluorescence image; Treat observing samples from the flashlight illumination that the broadband near-infrared light source of light source assembly sends, can obtain the optical coherence tomography image of sample after receiving by the interference signal probe assembly.
Principle of the present invention: cardinal principle of the present invention comprises that the burnt optical imagery of copolymerization is theoretical, fluorescence imaging is technological and Optical Coherence Tomography Imaging Technology.
The present invention compared with prior art has following advantage: the present invention is through associating laser co-focusing imaging technique and optical coherence tomography; Adopt same device to realize laser co-focusing imaging, fluorescence co-focusing imaging and optical coherent chromatographic imaging, can overcome the horizontal and lower shortcoming of axial resolution of the more shallow and single optical coherence tomography of single laser confocal microscope chromatography degree of depth system testing sample.
Description of drawings
Fig. 1 is the microscopical structural representation of multi-functional biomedical.
Fig. 2 is the components and parts detail list of system according to the invention.
Embodiment
According to Figure of description 1, the biomedical microscopical function that practical implementation the present invention proposes to how, introduce as follows in detail:
1, the illuminating bundle that is produced by the visible light source (1) of light source assembly gets into fibre-coupled mirrors head (5) through a road in the two-way output terminal of multi-channel optical fibre coupling mechanism (4); And get into two-dimensional scan assembly (8,9) after passing through catoptron (6) reflection and spectroscope (7) beam split successively.Pass through catoptron (10) reflection more successively, become times objective lens unit (11-13) focusing back lighting testing sample (14) from the illuminating laser beam of two-dimensional scan assembly (8,9) outgoing.
2, the visible light source of light source assembly (1) generally is a semiconductor laser; Be used to produce the laser beam of visible light wave range; Typical case's centre wavelength obtains higher optical system resolution thereby on sample, can form littler hot spot under this wave band about 405nm.
3, multi-channel optical fibre coupling mechanism (4) comprises a plurality of input ends and two output terminals, and it is exported according to the certain energy proportional distribution laser of multichannel input to two output terminals of coupling mechanism.Simultaneously, when echoed signal light during from any end input of its two output terminal, each input end of coupling mechanism also will oppositely be exported the echo laser that certain energy proportion distributes.
4, fibre-coupled mirrors head (5) can be an achromatism or aplanatic cemented doublet, also can be an aplanatic non-spherical lens, and it collimates to fiber coupler (4) output terminal emitting laser.Adopt the coupling camera lens (4) of different focal length can obtain the different collimated laser beam of beam diameter.
5, spectroscope (7) can be the spectroscope of plate, cube type or film-type, and it realizes the beam split function through the optical thin film of glass surface, and the light beam of miter angle incident is carried out beam split according to certain Transflective power ratio.In the practical implementation, spectroscope (7) also can be fixed on the runner, comprise dichroic beamsplitter one component light microscopic.Dichroic beamsplitter has the characteristic according to incident beam wavelength different decision beam reflection or transmission.Characteristic through the dichroic beamsplitter that the runner selection needs, makes fluorescent exciting reflect the illumination that realizes sample at the dichroic beamsplitter place in view of the above, and transmission realizes the detection to fluorescence at the dichroic beamsplitter place to make echo fluorescence.
6, two-dimensional scan assembly (8,9) comprises the optical scan vibration lens of two separate and quadratures.After the scanning of illuminating bundle through transversal scanning galvanometer (8); Form a laser focusing sweep trace on testing sample surface (14); Promptly realize wire illumination to testing sample (14); Pass through longitudinal scanning galvanometer (9) scanning again, form a laser focusing scanning plane, promptly realize face illumination testing sample (14) on testing sample surface (14).
7, becoming times objective lens unit (11-13) comprises lens 1 (11), lens 2 (12) and becomes times object lens (13).Lens 1 (11) are flat field telecentric scanning lens in typical case, and it assembles parallel beam, under the scan action of two-dimensional scan assembly (8,9), obtain the laser focusing of a face in the focal plane of lens 1 (11).Lens 3 (13) are far field chromatic aberration correction object lens, and it has higher numerical aperture and enlargement factor, can on sample, obtain as far as possible little hot spot on the one hand, collect the reflected light that sample produces on the other hand again as much as possible.Lens 2 (12) are the switching lens, and the light beam after it assembles lens 1 (11) is collimation again, thereby match the change times object lens (13) of far field chromatic aberration correction.Becoming times object lens (13) also can be the lens combination of one group of different numerical aperture and enlargement factor, during the practical implementation this patent, through rotation become times objective lens can select need change times object lens (13), to obtain different enlargement ratios and imaging viewing field.
8, the flashlight that reflection and scattering produce on change times objective lens unit (11-13) focal plane oppositely returns and after spectroscope (7) transmission beam split, is received by confocal acquisition of signal assembly (15-17) along illumination path.Horizontal and vertical scanning acquisition testing sample (14) through two-dimensional scan assembly (8,9) is cut image at the layer that becomes times objective lens unit (11-13) focal plane.Combine testing sample (14) to cut image with respect to the layer that the axially-movable that becomes times objective lens unit (11-13) obtains testing sample (14) different depth again, the burnt three-dimension layer of copolymerization that after Computerized three-dimensional reconstruct, can obtain testing sample (14) is cut image.
9, confocal acquisition of signal assembly (15-17) comprises collector lens (15), pin hole (16) and point probe (17).Wherein collector lens (15) focuses on echo beam; And be positioned at the interference that pin hole (16) on the focal plane of collector lens (15) is used to get rid of the horizontal and axial veiling glare of non-change times objective lens unit (11-13) along on the testing sample; Constitute confocal imaging system; Point probe (17) converts light signal into voltage signal for computer acquisition and processing, to obtain the reflection and the scattering properties of testing sample place laser focusing point.
10, collector lens (15) is two gummeds or non-spherical lens, and it focuses on the echoed signal light beam of spectroscope (7) transmission.The size of pin hole (16) is about laser forms the disperse hot spot at the sample place size, and this moment, system can obtain higher signal noise ratio level.And point probe (a 17) photomultiplier (PMT) or avalanche photo diode (APD) that has low noise amplifier normally, to realize detection and amplification under the low noise introducing condition to echoed signal.
11, the fluorescence excitation laser that is produced by the fluorescence light source (2) of light source assembly gets into fibre-coupled mirrors head (5) through a road in multi-channel optical fibre coupling mechanism (4) the two-way output terminal, and through catoptron (6), spectroscope (7) reflection back entering two-dimensional scan assembly (8,9).Pass through catoptron (10) reflection successively, become times objective lens unit (11-13) focusing back lighting testing sample (14) from the illuminating laser beam of two-dimensional scan assembly (8,9) outgoing.
12, realize after the scanning of fluorescence excitation laser process transversal scanning galvanometer (8) wire of testing sample (14) is thrown light on, pass through the scanning of longitudinal scanning galvanometer (9) again, realize face illumination testing sample (14).
13, the fluorescence light source of light source assembly (2) comprises several different wavelength of laser devices usually, and their type can be semiconductor laser, solid state laser, gas laser etc., and the typical wavelengths of their output has 405nm, 488nm etc.Can select different wavelengths or wavelength combinations to realize laser lighting through external circuit control laser instrument to the testing sample fluorescence excitation.
14, the exciting light and the generation of the fluorescence probe effect in the sample fluorescence that become on times objective lens unit (11-13) focal plane (perhaps do not have fluorescence probe in the sample; But under the laser excitation of suitable wavelength; Also there is fluorescence to produce; Be autofluorescence), oppositely return and after spectroscope (7) transmission beam split, receive along illumination path by confocal acquisition of signal assembly (15-17).Horizontal and vertical scanning acquisition testing sample (14) through two-dimensional scan assembly (8,9) is cut image at the layer that becomes times objective lens unit (11-13) focal plane.The layer that combines the axially-movable of testing sample (14) to obtain testing sample (14) different depth is again cut image, behind three-dimensionalreconstruction, can obtain the three-dimensional fluorescence image of testing sample (14).
15, the low coherent laser in broadband that is produced by the low relevant near-infrared laser light source (3) in the broadband of light source assembly through one in the output of multi-channel optical fibre coupling mechanism (4) two-way have a lot of social connections the low coherent laser of band as a reference light beam get into interference arm component (22-25); Through fibre-coupled mirrors head (22), mirror system (23) and chromatic dispersion coupling liquid (24); The back is reflected by catoptron (25), and returns multi-channel optical fibre coupling mechanism (4) along original optical path.
16, optical fiber coupled lens (22) is similar with fibre-coupled mirrors head (5), can be an achromatism or aplanatic cemented doublet, also can be an aplanatic non-spherical lens, and it collimates to fiber coupler (4) output terminal emitting laser.Adopt the coupling camera lens (4) of different focal length can obtain the different collimated laser beam of beam diameter.
17, mirror system (23) is made up of four catoptrons placing according to position as shown in the figure, and the distance of wherein top two-face mirror and following two-face mirror can be regulated, thereby can change the light path of interfering arm component (22-25).Chromatic dispersion matching fluid (24) is a kind of chemicals, mainly is used for the light beam of reference arm is carried out chromatic dispersion, in the hope of the dispersion characteristic of coupling from sample arm return signal light.
18, another in multi-channel optical fibre coupling mechanism (4) the two-way output terminal have a lot of social connections the low coherent laser of band as illuminating bundle through fibre-coupled mirrors head (5) collimation, and through catoptron (6), spectroscope (7) reflection back entering two-dimensional scan assembly (8,9).Pass through catoptron (10) reflection successively, become and realize illumination after times objective lens unit (11-13) focuses on from the illuminating laser beam of two-dimensional scan assembly (8,9) outgoing testing sample (14).
19, become on times objective lens unit (11-13) focal plane reflection and oppositely return multi-channel optical fibre coupling mechanism (4) along illumination path, with the broadband of interfering arm component (22-25) to return low coherent reference laser interference stack back entering interference signal probe assembly (18-21) with the flashlight that scattering produces.Reference arm assembly (22-25) through the adjustment mirror system (23) longitudinal separation with the coupling from reference arm reflected back flashlight and from sample than the light path between the flashlight that returns.Chromatic dispersion matching fluid (24) light beam of reference arm is carried out flashlight that chromatic dispersion reflects from reference arm with coupling and the flashlight that returns from sample arm between dispersion characteristics.
20, interference signal probe assembly (18-21) comprises coupled lens (18), diffraction grating (19), condenser lens (20) and linear array detector (21).Coupled lens (18) collimates to the echoed signal light that returns on the reference laser of interfering arm component (22-25) and returning and the testing sample; According to different wavelengths chromatic dispersion on different directions, the light beam after the chromatic dispersion passes through and is received by line array CCD (21) after collector lens (20) focuses on diffraction grating (19) again to light signal.Based on the low relevant lasing low coherence of near-infrared laser light source (3) in the broadband of light source assembly; The sequence light signal that light source assembly is received line array CCD (21) carries out spectrum analysis (being Fourier transform), obtains the reflection characteristic of each wave band of the interior different depth layer of testing sample (14).In conjunction with the two-dimensional scan of two-dimensional scan assembly (8,9), accomplish testing sample (14) 3-D view is extracted.
21, according to practical implementation step 1-10, the multi-functional biomedical microscope based on the imaging of optical coherence tomography and laser co-focusing of the present invention can obtain the confocal 3-D view of testing sample at detector terminal; According to practical implementation step 11-14, the multi-functional biomedical microscope based on the imaging of optical coherence tomography and laser co-focusing of the present invention can obtain the fluorescent confocal 3-D view of testing sample at detector terminal; According to practical implementation step 15-20, the multi-functional biomedical microscope based on the imaging of optical coherence tomography and laser co-focusing of the present invention can obtain the optical coherence tomography image of testing sample at detector terminal.
Through said process, can realize the abstraction function of high-resolution three-dimensional confocal images, three-dimensional fluorescence image and optical coherence tomography image to testing sample (14).
Need to prove; As above it is not restricted to listed utilization in instructions and the embodiment although preferable embodiment of the present invention is open, and it can be applied to various suitable the field of the invention fully; For being familiar with those skilled in the art; Can easily realize other modification, therefore under the universal that does not deviate from claim and equivalency range and limited, the legend that the present invention is not limited to specific details and illustrates here and describe.
Claims (5)
1. a multi-functional biomedical microscope that comprises laser scanning co-focusing imaging, fluorescence imaging and optical coherent chromatographic imaging is made up of light source assembly, two-dimensional scan assembly, change times objective lens unit, confocal acquisition of signal assembly, interference arm component and interference signal probe assembly.A kind of multi-functional biomedical microscope that this invention proposes; The visible light that utilizes light source assembly is as lighting source; Treat the observing samples surface through a two-dimensional scan assembly and a change times objective lens unit back lighting, the flashlight that reflects from sample surfaces is received by confocal acquisition of signal assembly, obtains the face scan image of sample; Layer through can obtain each degree of depth of sample to moving axially of sample is cut image, and the layer of each degree of depth is cut image obtains sample after rebuilding the burnt image of three-dimensional copolymerization; Treat observing samples from the light signal illumination that the fluorescence light source of light source assembly sends, the layer that obtains each degree of depth of sample is cut image and three-dimensional fluorescence image; The flashlight illumination testing sample that low relevant near-infrared light source sends from the broadband of light source assembly; By the optical coherence tomography image that can obtain sample after the reception of interference signal probe assembly; This invention is through introducing the burnt image of copolymerization that visible light source illumination sample obtains sample; Through introducing the fluoroscopic image that fluorescence light source illumination sample obtains sample; Through introducing the optical coherence tomography image that the low coherent laser light illumination sample in broadband obtains sample, realized a kind of functional biological medical science microscope, obtain the engineering three-dimensional tissue structures image on the transparent or nontransparent sample micro-scale.
2. according to the said multi-functional biomedical microscope of claim 1; It is characterized in that: said light source assembly comprises the low relevant near-infrared light source in visible light source, fluorescence light source and broadband; Visible light source is as beacon beam source lighting sample and obtain the burnt image of copolymerization of sample, and fluorescence LASER Light Source illumination sample obtains the fluorescent image; Near infrared broad band laser light illumination sample surfaces utilizes the interference signal probe assembly to obtain sample optical coherence tomography image.
3. according to the said multi-functional biomedical microscope of claim 1; It is characterized in that: said reference laser light arm component comprises coupled lens, catoptron, chromatic dispersion coupling liquid and reference light catoptron; Said chromatic dispersion matching fluid is a kind of chemicals; Mainly be used for the light beam of reference arm is carried out chromatic dispersion, in the hope of the dispersion characteristic of coupling from sample arm return signal light, said reference arm assembly changes from the optical path difference of reference arm reflected back flashlight through the position of adjustment reference mirror.
4. according to the said multi-functional biomedical microscope of claim 1, it is characterized in that: said two-dimensional scan assembly comprises horizontal galvanometer and vertical galvanometer, laterally galvanometer and the vertically plane of scanning motion mutually orthogonal of galvanometer; Laterally galvanometer high-velocity scanning; Its typical scan frequency is at 8kHz, vertical galvanometer low-velocity scanning, and its typical scan frequency is 20Hz; Through the synchronous scanning of two sides galvanometer, accomplish face scanning to testing sample.
5. according to the said multi-functional biomedical microscope of claim 1; It is characterized in that: said change times objective lens unit; Comprise lens 1 and realize beacon beam scanning, comprise lens 2 and realize the beacon beam coupling, comprise change times object lens and realize optically focused observation testing sample; Said change times object lens are the short focus objective lens of high power, and its typical focal length is 10mm.
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| CN108427207A (en) * | 2017-12-31 | 2018-08-21 | 浙江红谱科技有限公司 | Infrared image emerging system and method with instruction function |
| CN108387519A (en) * | 2018-05-03 | 2018-08-10 | 上海市质子重离子临床技术研发中心 | Microscopic system is just set in up-conversion luminescence near-infrared and the multi-functional wide field of colour imaging |
| CN113520303A (en) * | 2021-07-15 | 2021-10-22 | 苏州微清医疗器械有限公司 | Method for improving resolution of confocal scanning optical imaging image |
| CN114322797A (en) * | 2021-12-31 | 2022-04-12 | 浙江大学嘉兴研究院 | Optical detection cutting method and system for biological membrane tissue based on weak coherent interference |
| CN117250743A (en) * | 2023-11-16 | 2023-12-19 | 北京攸维医疗科技有限公司 | Multi-mode imaging method and device |
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