CN202814861U - Multifunctional biomedical microscope - Google Patents

Abstract

The utility model relates to a multifunctional biomedical microscope having the functions of laser scanning confocal imaging, fluorescence imaging and optical coherence chromatography imaging. The multifunctional biomedical microscope is composed of a light source component, a two-dimensional scanning component, a variable lens component, a confocal signal detecting component, a coherence arm component and a coherence signal detecting component. According to the utility model, confocal images of samples are obtained through introducing a visible light source to illuminate samples and the fluorescent images of the samples are obtained through introducing a fluorescent light source to illuminate the samples; and optical coherence chromatography images of the samples are obtained through introducing a wide band low-coherence near infrared laser light source to illuminate samples. The multifunctional biomedical microscope is realized so as to obtain three-dimensional tissue structure images of transparent or nontransparent samples on microscale.

Description

A kind of multi-functional biomedical microscope

Technical field

The present invention relates to a kind of multi-functional biomedical microscope, particularly a kind of for the biomedical tissue imaging, and can obtain the multi-functional biomedical microscope of Confocal Images, fluoroscopic image and optical coherence tomography image.

Background technology

The concept 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, the multidigit scholars such as A.F.Slomba and C.J.R.Sheppard have carried out finer research to confocal imaging.Laser confocal microscope system, the confocal fluorescent microscopic system of combined with fluorescent probe Detection Techniques particularly, because the employing of detecting pinhole and shorter wavelength laser is so that system has higher lateral resolution (about 100nm) and longitudinal frame (about 50nm), be 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. 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 large order of magnitude of Laser Scanning Confocal Microscope.But the finite bandwidth of low-coherence light source has limited its longitudinal frame, and the simultaneously employing of long wavelength laser has also reduced the lateral resolution of system.

In sum as can be known, 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, and the two all demands urgently improving.

Contrast international and domestic technological achievement in confocal microscopic image 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, realization is to the laser confocal imaging of testing sample, fluorescence imaging and optical coherent chromatographic imaging.

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.

Technical solution of the present invention:

A kind of multi-functional biomedical microscope is made of light source assembly, two-dimensional scan assembly, zoom objective assembly, confocal acquisition of signal assembly, interference arm component and interference signal probe assembly.Utilize the visible light of light source assembly as lighting source, by two-dimensional scan assembly and zoom objective assembly back lighting sample surfaces to be observed, 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 by 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; The light signal that sends from the fluorescence light source of the light source assembly sample to be observed that throws light on, the layer that can obtain each degree of depth of sample is cut image and three-dimensional fluorescence image; The flashlight that sends from the broadband near-infrared light source of the light source assembly sample to be observed that throws light on, by the optical coherence tomography image that can obtain sample after the reception of interference signal probe assembly, realize a kind of functional biological medical science microscope, obtained the engineering three-dimensional tissue structures image on the transparent or nontransparent sample micro-scale.

Principle of the present invention: cardinal principle of the present invention comprises the burnt optical imagery theory of copolymerization, Imaging-PAM and Optical Coherence Tomography Imaging Technology.

The present invention compared with prior art has the following advantages: the present invention is by associating laser confocal imaging technology and optical coherence tomography, adopt same device realization to laser confocal imaging, fluorescence co-focusing imaging and the optical coherent chromatographic imaging of testing sample, can overcome the more shallow and single Optical coherence tomography of the single laser confocal microscope chromatography degree of depth laterally and the lower shortcoming of axial resolution.

Description of drawings

Fig. 1 is the microscopical structural representation of multi-functional biomedical.

Table 1 is the components and parts detail list of system of the present invention.

Table 1

1, visible light source	2, fluorescence light source	3, near-infrared light source	4, fiber coupler	5, coupled lens
					6, plane mirror	7,spectroscope	8, horizontal galvanometer	9,vertical galvanometer	10,plane mirror
11,lens 1	12, lens 2	13,zoom objective	14,testing sample	15,collector lens
					16,pin hole	17,point probe	18, coupledlens	19,diffraction grating	20,collector lens
21,linear array detector	22, coupledlens	23,catoptron group	24, dispersion liquid	25, plane mirror

Embodiment

According to Figure ofdescription 1, the biomedical microscopical function that implementation the present invention proposes to how is described in detail as follows:

1, the illuminating bundle that is produced by the visible light source (1) of light source assembly enters coupling fiber camera lens (5) by a road in the two-way output terminal of multi-channel optical fibre coupling mechanism (4), and enter two-dimensional scan assembly (8,9) after passing through successively catoptron (6) reflection and spectroscope (7) light splitting.Pass through successively again catoptron (10) reflection, zoom objective assembly (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 semiconductor laser, laser beam for generation of visible light wave range, typical case's centre wavelength is about 405nm, thereby on sample, can form less hot spot under this wave band and obtain higher optical system resolution, as beacon beam source lighting sample and obtain the Confocal Images of sample.

3, multi-channel optical fibre coupling mechanism (4) comprises a plurality of input ends and two output terminals, and it is assigned to the laser of multichannel input two output terminal outputs of coupling mechanism according to certain energy proportion.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, coupling fiber camera lens (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, Cubic or film-type, and it realizes the light splitting function by the optical thin film of glass surface, and the light beam of miter angle incident is carried out light splitting according to certain Transflective power ratio.In the implementation, spectroscope (7) also can be fixed on the runner, comprise dichroic beamsplitter one component light microscopic.Dichroic beamsplitter has according to the different characteristics that determine beam reflection or transmission of incident beam wavelength.Characteristic by the dichroic beamsplitter that the runner selection needs, makes fluorescent exciting in the illumination of dichroic beamsplitter place reflection realization to sample accordingly, makes echo fluorescence in the detection of transmission realization in dichroic beamsplitter place to fluorescence.

6, two-dimensional scan assembly (8,9) comprise the optical scan vibration lens of two separate and quadratures, be horizontal galvanometer (8) and vertical galvanometer (9), both planes of scanning motion are mutually orthogonal, laterally galvanometer (8) high-velocity scanning, its typical scan frequency is at 8kHz, vertical galvanometer (9) low-velocity scanning, its typical scan frequency is 20Hz, after the scanning of illuminating bundle through transversal scanning galvanometer (8), form a laser focusing sweep trace on testing sample surface (14), namely realize the wire illumination to testing sample (14), pass through again longitudinal scanning galvanometer (9) scanning, form a laser focusing scanning plane on testing sample surface (14), namely realize the face illumination to testing sample (14).

7, zoom objective assembly (11-13) comprises lens one (11), lens two (12) and zoom objective (13).Lens one (11) are flat field telecentric scanning lens in typical case, 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 one (11), realize beacon beam scanning.Zoom objective (13) is far field chromatic aberration correction object lens, it has higher numerical aperture and enlargement factor, can obtain as far as possible little hot spot at sample on the one hand, collect as much as possible again on the other hand the reflected light that sample produces, realize the optically focused observation to testing sample.Lens two (12) are the switching lens, and the light beam after it assembles lens 1 (11) collimates again, thereby match the zoom objective (13) of far field chromatic aberration correction, realize the beacon beam coupling.Zoom objective (13) also can be the lens combination of one group of different numerical aperture and enlargement factor, during the implementation this patent, can select the zoom objective (13) of needs by rotating the zoom objective group, to obtain different enlargement ratios and imaging viewing field.

8, the flashlight that reflection and scattering produce on zoom objective assembly (11-13) focal plane oppositely returns and after spectroscope (7) transmission light splitting, is received by confocal acquisition of signal assembly (15-17) along illumination path.Horizontal and vertical scanning acquisition testing sample (14) by two-dimensional scan assembly (8,9) is cut image at the layer of zoom objective assembly (11-13) focal plane.Cut image in conjunction with testing sample (14) with respect to the layer that the axially-movable of zoom objective assembly (11-13) obtains testing sample (14) different depth again, the burnt three-dimension layer of copolymerization that can obtain as calculated testing sample (14) behind the machine three-dimensionalreconstruction 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 for getting rid of the horizontal and axial veiling glare at non-zoom objective assembly (11-13) focus place on the testing sample, consist of confocal imaging system, point probe (17) is converted to voltage signal for computer acquisition and processing with light signal, to obtain 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 (17) normally a photomultiplier with low noise amplifier (PMT) or avalanche photodide (APD), to realize detection and the amplification to echoed signal under the low noise introducing condition.

11, the fluorescence excitation laser that is produced by the fluorescence light source (2) of light source assembly enters coupling fiber camera lens (5) by a road in multi-channel optical fibre coupling mechanism (4) the two-way output terminal, and after catoptron (6), spectroscope (7) reflection, enter two-dimensional scan assembly (8,9).Pass through successively catoptron (10) reflection, zoom objective assembly (11-13) focusing back lighting testing sample (14) from the illuminating laser beam of two-dimensional scan assembly (8,9) outgoing.

12, fluorescence excitation laser is realized the wire of testing sample (14) is thrown light on through the scanning of transversal scanning galvanometer (8) afterwards, passes through the scanning of longitudinal scanning galvanometer (9) again, realizes the face illumination to testing sample (14).

13, the fluorescence light source of light source assembly (2) comprises the laser instrument of several different wave lengths 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 wavelength or wavelength combinations realization to the laser lighting of testing sample fluorescence excitation by external circuit control laser instrument, obtain the fluorescent image.

14, the exciting light on zoom objective assembly (11-13) focal plane and the fluorescence probe effect in the sample produce fluorescence (does not perhaps 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 light splitting, received by confocal acquisition of signal assembly (15-17) along illumination path.Horizontal and vertical scanning acquisition testing sample (14) by two-dimensional scan assembly (8,9) is cut image at the layer of zoom objective assembly (11-13) focal plane.The layer that obtains testing sample (14) different depth in conjunction with the axially-movable of testing sample (14) is again cut image, can obtain the three-dimensional fluorescence image of testing sample (14) behind three-dimensionalreconstruction.

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 enters interference arm component (22-25) by the low coherent laser of band of having a lot of social connections of one in the output of multi-channel optical fibre coupling mechanism (4) two-way as the reference light beam, through coupling fiber camera lens (22), mirror system (23) and dispersion coupling liquid (24), reflected by catoptron (25) afterwards, and return multi-channel optical fibre coupling mechanism (4) along original optical path, near infrared broad band laser light illumination sample surfaces utilizes the interference signal probe assembly to obtain sample optical coherence tomography image.

16, coupling fiber lens (22) are similar with coupling fiber camera lens (5), can be an achromatism or aplanatic cemented doublet, also can be an aplanatic non-spherical lens, 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 comprised of four catoptrons according to as shown in the figure position placement, wherein the distance of top two-face mirror and following two-face mirror can be regulated, thereby can change the optical path difference of interfering arm component (22-25) to be reflected back flashlight.Dispersion matching fluid (24) is a kind of chemicals, is mainly used to the light beam of reference arm is carried out 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 had a lot of social connections and is with low coherent laser to collimate through coupling fiber camera lens (5) as illuminating bundle, and after catoptron (6), spectroscope (7) reflection, enter two-dimensional scan assembly (8,9).Pass through successively catoptron (10) reflection, the rear illumination that realizes testing sample (14) of zoom objective assembly (11-13) focusing from the illuminating laser beam of two-dimensional scan assembly (8,9) outgoing.

19, the flashlight that reflection and scattering produce on zoom objective assembly (11-13) focal plane oppositely returns multi-channel optical fibre coupling mechanism (4) along illumination path, enters interference signal probe assembly (18-21) after superposeing with the low coherent reference laser interference in the broadband of interfering arm component (22-25) to return.The longitudinal separation of reference arm assembly (22-25) by adjusting mirror system (23) with coupling from reference arm be reflected back flashlight and from sample than the light path between the flashlight that returns.Dispersion matching fluid (24) light beam of reference arm is carried out flashlight that 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 the dispersion on different directions of different wavelength, the light beam after the 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), finish testing sample (14) 3-D view is extracted.

21, according to implementation step 1-10, the multi-functional biomedical microscope based on optical coherence tomography and laser confocal imaging of the present invention can obtain at detector terminal the confocal 3-D view of testing sample; According to implementation step 11-14, the multi-functional biomedical microscope based on optical coherence tomography and laser confocal imaging of the present invention can obtain at detector terminal the fluorescent confocal 3-D view of testing sample; According to implementation step 15-20, the multi-functional biomedical microscope based on optical coherence tomography and laser confocal imaging of the present invention can obtain at detector terminal the optical coherence tomography image of testing sample.

Through said process, can realize to testing sample (14) abstraction function of high-resolution three-dimensional confocal images, three-dimensional fluorescence image and optical coherence tomography image.

Need to prove, although better embodiment of the present invention is open as above, but it is not restricted to listed utilization in instructions and the embodiment, it can be applied to various suitable the field of the invention fully, for those skilled in the art, therefore can easily realize other modification, not deviate under the universal that claim and equivalency range limit that the present invention is not limited to specific details and illustrates and the legend of describing here.

Claims (5)

1. multi-functional biomedical microscope, it is characterized in that: by light source assembly, the two-dimensional scan assembly, the zoom objective assembly, confocal acquisition of signal assembly, interfere arm component and interference signal probe assembly to consist of, utilize the visible light of light source assembly as lighting source, by two-dimensional scan assembly and zoom objective assembly back lighting sample surfaces to be observed, the flashlight that reflects from sample surfaces is received by confocal acquisition of signal assembly, obtain the face scan image of sample, cut image by the layer that can obtain each degree of depth of sample to moving axially of sample, the layer of each degree of depth is cut image obtains sample after rebuilding three-dimensional Confocal Images, the light signal that sends from the fluorescence light source of the light source assembly sample to be observed that throws light on, 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 obtains the Confocal Images of sample by introducing visible light source illumination sample, obtain the fluoroscopic image of sample by introducing fluorescence light source illumination sample, obtain the optical coherence tomography image of sample by introducing the low coherent laser light illumination sample in broadband, realize a kind of functional biological medical science microscope, obtained the engineering three-dimensional tissue structures image on the transparent or nontransparent sample micro-scale.

2. described multi-functional biomedical microscope according to claim 1, it is characterized in that: described 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 Confocal Images of sample, fluorescence LASER Light Source illumination sample, obtain 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. described multi-functional biomedical microscope according to claim 1, it is characterized in that: described interference arm component comprises coupled lens, catoptron, dispersion coupling liquid and reference light catoptron, described dispersion matching fluid is a kind of chemicals, be mainly used to the light beam of interfering arm is carried out dispersion, in the hope of the dispersion characteristic of coupling from sample return signal light, described interference arm component changes from interfering arm to be reflected back the optical path difference of flashlight by the position of adjusting the reference light catoptron.

4. described multi-functional biomedical microscope according to claim 1, it is characterized in that: described two-dimensional scan assembly comprises horizontal galvanometer and vertical galvanometer, laterally galvanometer is mutually orthogonal with the plane of scanning motion of vertical 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, by the synchronous scanning of two sides galvanometer, finish the face scanning to testing sample.

5. described multi-functional biomedical microscope according to claim 1 is characterized in that: described zoom objective assembly, and comprise lens one and realize beacon beam scanning, comprise lens two and realize the beacon beams coupling, comprise zoom objective and realize optically focused observation to testing sample.

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