CN212514263U

CN212514263U - Hyperspectral imaging microscope

Info

Publication number: CN212514263U
Application number: CN202020567608.3U
Authority: CN
Inventors: 王懋; 吴东岷; 范娜娜; 汪远
Original assignee: Nanjing Weina Technology Research Institute Co ltd
Current assignee: Nanjing Weina Shijie Medical Technology Co.,Ltd.
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2021-02-09
Anticipated expiration: 2030-04-16

Abstract

The utility model provides a hyperspectral imaging microscope, which comprises an excitation light source, an objective table, an objective group, a Kohler optical lens group, a spectroscope and a convergent lens; the objective table reflects part of the light emitted by the excitation light source and sequentially penetrates through the objective lens group, the spectroscope, the convergent lens, the liquid crystal tunable filter and the area array photoelectric detector. The microscope adopts the liquid crystal tunable filter, adapts to reflected light and fluorescence of samples under different wavelengths, has high-resolution reflection imaging and fluorescence imaging, and does not need to manufacture a plurality of narrow and wide filter plates; in addition, the microscope adopts a single-wavelength laser as an excitation light source, has more photons, and enables a sample to have more obvious reflected light.

Description

Hyperspectral imaging microscope

Technical Field

The utility model relates to a biomedical field, concretely relates to high spectrum imaging microscope and imaging method.

Background

The spectral imaging technology is a powerful means for researching life mechanism in the field of observing cell activities, is particularly applied to cell detection, and can play a role in identifying different cells, distinguishing different growth stages of the cells, analyzing certain elements in the cells, detecting the curative effect of drugs after cell administration and the like. With the continuous development and improvement of the spectral imaging technology, the spectral imaging technology will play a greater role in the biomedical field, and in the aspect of microscopic imaging, the spectral imaging technology provides powerful support for deeply researching various structures and characteristics of microstructures.

Reflectance imaging can detect local changes in tissue scattering and absorption properties, and fluorescence imaging can detect changes in histochemical composition.

A part of the light absorbed by the biological tissue is converted into heat energy, and the other part is used for exciting fluorescence and phosphorescence. Incident light in the ultraviolet or near-infrared band excites tissue molecules and induces them to emit fluorescence, most fluorophores are associated with tissue structural matrices and cellular metabolic pathways. The most common fluorophores in biological tissue matrices are collagen and elastin, while the primary fluorophores involved in cellular metabolism are Nicotinamide Adenine Dinucleotide (NADH), Flavin Adenine Dinucleotide (FAD) and liposomes. These fluorophores have different fluorescence intensities in different spectral ranges. For example, the bandwidth of fluorescence generated by collagen or elastin under the irradiation of light waves of 400-1700nm is higher than that of light of 400-600nm, and the difference can be used for distinguishing different types of tissues, such as epithelial tissues and connective tissues. Cells with different degrees of pathology have different structures and metabolic rates, and therefore their emitted fluorescence spectra also differ. Therefore, without using a fluorescent agent, it is possible to detect tissues in real time by fluorescence imaging and to perform diagnosis of diseases.

Conventional imaging is for example: fluorescence spectral imaging and raman spectral imaging have a considerable spectrum detection capability, but the fluorescence spectral imaging can only realize spectral imaging under certain discrete wavelengths (under specific partial wavelengths), the information content is insufficient, deep research and analysis on a sample are not facilitated, the raman spectral imaging is used as a nonlinear optical phenomenon, the efficiency of raman scattering is very low, only one photon can generate frequency drift through the raman effect in 105 photons, the frequency drift is difficult to observe in common illumination, and a laser with high photon density is used as an excitation light source.

SUMMERY OF THE UTILITY MODEL

In order to overcome the defects in the prior art, the utility model provides a hyperspectral imaging microscope, which comprises an excitation light source, an objective table, an objective lens group, a Kohler optical lens group, a spectroscope and a convergent lens and is characterized in that,

in order to achieve the purpose, light emitted by the excitation light source sequentially penetrates through the Kohler optical lens group, the spectroscope and the objective lens group and irradiates on the objective table, one side, away from the objective table, of the spectroscope is sequentially provided with the convergent lens, the liquid crystal tunable filter and the area array photoelectric detector, and the objective table reflects part of the light emitted by the excitation light source and sequentially penetrates through the objective lens group, the spectroscope, the convergent lens, the liquid crystal tunable filter and the area array photoelectric detector.

Furthermore, the light-transmitting wave band of the liquid crystal tunable filter is continuously adjustable.

Further, the area array photoelectric detector comprises an EMCCD camera, a CCD camera, a coms camera, a scoms camera and an InGaAs camera.

Furthermore, the resolution of the objective lens group is adjustable.

Further, the excitation light source includes one or more lasers of different wavelengths.

The utility model discloses still provide a hyperspectral imaging method for implementing above-mentioned hyperspectral imaging microscope and design, including following step: s1: the sample on the objective table is irradiated by light sources with different wavelengths, the sample emits fluorescence and reflects part of excitation light source under the stimulation of the light sources, and the fluorescence and the reflected light sequentially pass through the objective lens group, the spectroscope and the convergent lens to reach the liquid crystal tunable filter;

s2: adjusting the light transmission wave band of the liquid crystal tunable filter, so that fluorescence and reflected light generated by the sample under the irradiation of light sources with different wavelengths are transmitted to the surface of the area array photoelectric detector;

s3: the excited fluorescence or reflected light is converged on the area array photoelectric detector, and a reflected hyperspectral image and a fluorescence hyperspectral image of the sample can be scanned;

s4: the area array photoelectric detector converts the received optical signals into electric signals and transmits the electric signals to the computer, and the computer processes the received data and displays the reflected hyperspectral image and the fluorescence hyperspectral image of the sample.

Has the advantages that: 1. the utility model adopts the liquid crystal tunable filter, the light transmission wave band of which can be continuously adjusted, and the filter can adapt to the reflected light and fluorescence of samples under different wavelengths, especially the fluorescence of samples in the wavelength ranges of near infrared and visible light, thereby having the reflected imaging and fluorescence imaging with higher resolution, and needing no manufacture of a plurality of narrow and wide filter plates;

2. the utility model discloses a single or a plurality of laser instrument constitution excitation light source, can arouse fluorescence wide range, have more photon, can make the sample have comparatively obvious reverberation.

Drawings

The invention will be further described and illustrated with reference to the accompanying drawings.

Fig. 1 is a schematic structural view of the whole of the present invention;

fig. 2 is a flow chart of the present invention.

Reference numerals: 1. an excitation light source; 2. a Kohler optical lens; 3. a beam splitter; 4. an objective lens group; 5. an object stage; 6. a sample; 7. a converging lens; 8. a liquid crystal tunable filter; 9. an area array photodetector; 10. and (4) a computer.

Detailed Description

The technical solution of the present invention will be more clearly and completely explained by the description of the preferred embodiments of the present invention with reference to the accompanying drawings.

Examples

The utility model provides a high spectrum imaging microscope, including objective table 5, objective group 4, spectroscope 3 and convergent lens 7, excitation light source 1, the optical lens 2 groups of kohler, liquid crystal tunable filter 8 and area array photoelectric detector 9.

As shown in fig. 1, a specimen 6 (e.g., a cytological slide) is placed on a stage 5, an excitation light source 1 simultaneously emits light of a certain wavelength, the light is irradiated on a spectroscope 3 through a kohler optical lens 2 set, the spectroscope 3 reflects the light, and the reflected light is irradiated on the specimen 6 through an objective lens set 4 and the stage 5 in this order. The sample 6 excites fluorescence under the illumination of the excitation light source 1, and simultaneously the sample 6 reflects part of the light of the excitation light source 1 with single wavelength (hereinafter referred to as reflected light), and the fluorescence and the reflected light sequentially pass through the objective table 5, the objective lens group 4, the spectroscope 3, the converging lens 7 and the liquid crystal tunable filter 8. The liquid crystal tunable filter 8 performs two-dimensional spatial light splitting of the fluorescence and the reflected light by adjusting the light transmission wavelength band.

The area array photoelectric detector 9 arranged at the tail end of the light path of the liquid crystal tunable filter 8 receives and displays the fluorescence or reflected light after two-dimensional space light splitting step by step, and the displayed images are respectively a reflected hyperspectral image and a fluorescence hyperspectral image.

After the area array photoelectric detector 9 collects primary fluorescence and reflected light, the excitation light source 1 is closed and restarted, the excitation light source 1 irradiates the sample 6 again by using light with another wavelength different from the wavelength, and the sample 6 presents a reflected hyperspectral image and a fluorescence hyperspectral image on the area array photoelectric detector 9 again through the process.

As shown in fig. 1, the laser light source comprises one or more lasers with different wavelengths, each of the one or more lasers with different wavelengths is, for example, a type BOT980-5000D laser, the wavelength range of the emitted photons of the excitation light source includes a part of visible light and a part of infrared light, and the output laser wavelengths include 405nm, 488nm, 561nm, 640nm, 785nm, 808nm, 980nm, 1064nm, etc. The emission light of the excitation light source has a plurality of photons;

the utility model adopts the above laser light source, it is more through the wavelength that sends laser, overcome the not enough shortcoming of the information content that adopts discrete wavelength (under specific part wavelength) sample 6 formation of image among the prior art, excitation light source 1 has more wavelength selection, can obtain reflection hyperspectral image and fluorescence hyperspectral image under the different wavelength, and the sample 6 formation of image information content that obtains is more complete. Meanwhile, the excitation light source 1 is adopted, so that more photons are provided, and the sample 6 can have more obvious reflected light.

The liquid crystal tunable filter 8 adopts a tunable filter with the model number of KURIIOS _ WL1, the liquid crystal tunable filter is a device manufactured according to the liquid crystal electric control birefringence effect, has the function of continuously adjusting the light transmitting wave band, and is used for filtering the excitation light source and the interference light outside the other excitation fluorescence wave bands, and the fluorescence emitted by the sample 6 is imaged on the high-sensitivity area array photoelectric detector 9 by the imaging objective lens after passing through the liquid crystal tunable filter. Because the laser wavelength range emitted by the laser is wide, the adoption of the continuously adjustable liquid crystal tunable filter 8 can adapt to the filtering of light with different wavelengths.

As shown in fig. 1, the high-sensitivity area-array photodetector 9 is a camera set mainly composed of an EMCCD (electron multiplying CCD) camera, a CCD camera, a coms (complementary metal oxide conductor device) camera, a scoms camera, an InGaAs camera, and the like. The high-sensitivity area array photoelectric detector 9 is used for receiving a plurality of images of the sample 6 transmitted by the liquid crystal tunable filter 8 under different wavelengths of the excitation light source 1, and then artificially selecting the image of the sample 6 with high resolution from the plurality of images. In order to match with the high-sensitivity area array photoelectric detector 9 and adapt to the imaging high resolution of the sample 6 under the wavelength of different single-wavelength excitation light sources 1, the objective lens also has the function of adjustable resolution, so that the objective lens has high light transmittance.

The excitation light source 1, the liquid crystal tunable filter 8 and the area array photoelectric detector 9 are electrically connected with a computer 10 through a communication interface (such as a USB3.0 interface), the computer 10 can control the start and stop of the excitation light source 1, the computer 10 can also adjust the light transmission waveband of the liquid crystal tunable filter 8, and the fluorescence hyperspectral image or the reflection hyperspectral image detected by the area array photoelectric detector 9 can be displayed.

As shown in fig. 2, the utility model discloses still provide a hyperspectral imaging method that is used for above-mentioned hyperspectral imaging microscope to design, include following step:

s1: the sample on the objective table is irradiated by light sources with different wavelengths, the sample emits fluorescence and reflects part of excitation light source under the stimulation of the light sources, and the fluorescence and the reflected light sequentially pass through the objective lens group, the spectroscope and the convergent lens to reach the liquid crystal tunable filter;

s2: adjusting the light transmission wave band of the liquid crystal adjustable filter, so that fluorescence and reflected light generated by the sample under the irradiation of light sources with different wavelengths are transmitted to the surface of the area array photoelectric detector;

s3: the excited fluorescence or reflected light is converged on an area array photoelectric detector, and a reflected hyperspectral image and a fluorescence hyperspectral image of the sample can be scanned;

The utility model discloses the benefit that still possesses has:

1. the liquid crystal tunable filter is adopted, and has the advantages of high switching speed of the transmission waveband range, high spectrum scanning speed, simple operation, narrow gating wavelength band, high spectrum resolution, no moving part, no mechanical jitter, large aperture, large field angle, small volume and good optical characteristic;

2. by adopting a hyperspectral imaging technology, overlapping fluorescence spectrum regions are separated through an algorithm by a narrow-band filter liquid crystal tunable filter, so that fluorescence spectrum separation is realized, and accurate quantitative analysis is carried out on a fluorescence marker;

3. the microobjective has 4X \10X \20X \40X \50X \60X with high light transmittance, high NA value and high imaging resolution, and simultaneously needs to be matched with a corresponding area array photoelectric detector, and the magnification of the objective can be switched;

4. the liquid crystal can modulate the wave band to be wide and continuously adjustable, and the system is suitable for various biological fluorescence, fluorescent probes and nano materials. In addition, for multiple fluorescence markers in a sample body, an excitation light source is rapidly switched, and the light transmission wave band of the liquid crystal tunable filter is adjusted, so that the excited fluorescence can be converged on the area array photoelectric detector by the imaging objective lens through the liquid crystal tunable filter, and a hyperspectral image of the sample can be scanned;

5. the microscope body adopts an upright fluorescence microscope or an inverted fluorescence microscope lens. The above detailed description merely describes the preferred embodiments of the present invention and does not limit the scope of the present invention. Without departing from the design concept and spirit scope of the present invention, the ordinary skilled in the art should belong to the protection scope of the present invention according to the present invention provides the text description and drawings to the various modifications, replacements and improvements made by the technical solution of the present invention. The scope of protection of the present invention is determined by the claims.

Claims

1. The hyperspectral imaging microscope comprises an excitation light source, an objective table, an objective group, a Kohler optical lens group, a spectroscope and a convergent lens,

the device comprises an excitation light source, a objective table, a Kohler optical lens group, a spectroscope and an objective lens group, wherein light emitted by the excitation light source sequentially penetrates through the Kohler optical lens group, the spectroscope and the objective lens group and irradiates on the objective table, a converging lens, a liquid crystal tunable filter and an area array photoelectric detector are sequentially arranged on one side of the spectroscope, which is far away from the objective table, and part of light emitted by the excitation light source is reflected by the objective table and sequentially penetrates through the objective lens group.

2. The hyperspectral imaging microscope according to claim 1, wherein the light transmission band of the liquid crystal tunable filter is continuously adjustable.

3. The hyperspectral imaging microscope of claim 1, wherein the area array photodetector comprises an EMCCD camera, a CCD camera, a coms camera, a scoms camera, an InGaAs camera.

4. The hyperspectral imaging microscope of claim 1, wherein the resolution of the objective lens group is tunable.

5. The hyperspectral imaging microscope of claim 1, wherein the excitation light source comprises one or more lasers of different wavelengths.

6. The hyperspectral imaging microscope of claim 5, wherein the wavelength range of the emitted photons of the laser comprises part of visible light and part of infrared light, and further wherein the wavelength of the light emitted by the excitation light source comprises 405nm, 488nm, 561nm, 640nm, 785nm, 808nm, 980nm, 1064 nm.