CN212159566U

CN212159566U - Hyperspectral living body fluorescence molecule imaging system

Info

Publication number: CN212159566U
Application number: CN202020569949.4U
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: 2020-12-15
Anticipated expiration: 2030-04-16

Abstract

The utility model discloses a hyperspectral live body fluorescence molecule imaging system is including the objective table that is used for bearing the weight of the sample and the computer that is used for control to handle, the objective table is arranged in the darkroom box, be equipped with lighting system and life support system on the objective table, the top of objective table is equipped with the light source subassembly, the objective table top still is equipped with the receiving arrangement who is used for receiving the sample and arouses back emission fluorescence, receiving arrangement is including the filter equipment who arranges in proper order, imaging lens, area array photoelectric detector and image collector, filter equipment is liquid crystal tunable filter or filter group, receiving arrangement can fast switch over its printing opacity wave band in order to receive the printing opacity wave band in by the fluorescence of arousing the back emission, carry out fluorescence imaging to the sample, the spectral information through the fluorescence of quick acquisition every wave band arouses simultaneously realizes hyperspectral. The system has continuously adjustable filtering wavelength and can improve spectral imaging resolution.

Description

Hyperspectral living body fluorescence molecule imaging system

Technical Field

The utility model relates to a high spectrum live body fluorescence molecule imaging system belongs to optics technical field.

Background

In the field of biological detection, spectral imaging techniques are mainly applied to the detection of biological materials, biological tissues, biological cells, biological molecules, and the like. For biological materials and tissues in general, analysis can be directly performed by detecting reflection or absorption spectra due to differences in reflection or absorption spectra of different materials or tissues. However, in the case of a detection object having a relatively low contrast such as a biological cell and a relatively small size such as a biological molecule, the detection object cannot be directly analyzed and processed by a reflection or absorption spectrum of the detection target. Other approaches, such as fluorescent probes, nanomaterials, etc., are needed to indirectly acquire biological information for detection of single or multiple targets for further processing and analysis.

The living body fluorescence imaging system is mainly applied to qualitative, quantitative and positioning research of tissue, cell and even molecular level behaviors in a biological process under a living body state, wherein the fluorescence imaging is particularly suitable for living body imaging of viruses with specific optical molecular markers through gene modification or chemical modification. The biological tissue can emit fluorescence after being excited by light with specific wavelength, and in abnormal development and canceration areas, the blue-green autofluorescence of the biological tissue has a tendency of attenuation, and the red autofluorescence has a tendency of enhancement, so that the autofluorescence imaging method of the biological tissue can be used as a sensitive and simple lesion detection way. Although autofluorescence of biological tissues can be used for detection and diagnosis of certain diseases, the complexity and relatively weak signal level of autofluorescence has so far prevented its widespread use in the biological and medical fields. In the microscopic field, since the size of biomolecules is very small, such as protein molecules, nucleic acid molecules, tumor molecules, etc., generally between several nanometers and several tens of nanometers, which is smaller than the wavelength range of light, the scattering and absorption effect of light is not obvious, and thus the biomolecules cannot be directly detected by spectroscopy. While a fluorescent probe may absorb photons of a particular frequency, thereby exciting photons of another frequency. By detecting the newly generated photons, the presence of the fluorescent probe can be detected. Therefore, fluorescent probes and nanomaterials are widely used in the field of biomolecule detection.

However, the current living body fluorescence imaging device has a plurality of defects, such as: the method has the defects of less spectral channels, longer time for switching the spectral channels, defocusing caused by mechanical vibration when the optical filter is mechanically switched, large volume and the like, and cannot meet the increasing requirements of high-precision, high-resolution and multi-component biological detection.

Disclosure of Invention

For overcoming the not enough of prior art, the utility model provides a high spectrum live body fluorescence molecule imaging system can obtain high spectral image in live body fluorescence imaging system to filter passes through the optical wavelength continuously adjustable, can realize multispectral fluorescence separation, realizes the two-dimentional or three-dimensional formation of image of quick collection every spectrum, can improve spectral imaging resolution.

In order to achieve the above object, the present invention provides a hyperspectral living body fluorescence molecule imaging system, which comprises an object stage for bearing a sample, a computer for controlling the system and processing information, wherein the object stage is arranged in a darkroom box, the object stage is further provided with an illumination system and a life support system, a light source assembly for generating excitation light is arranged above the object stage, the light source assembly comprises one or more single-wavelength exciter groups and a light source homogenization module, a receiving device for receiving fluorescence emitted by the sample after being excited is further arranged above the object stage, the receiving device comprises a filter device, an imaging lens, an area array photodetector and an image collector which are arranged in sequence, the filter device is a liquid crystal tunable filter or a filter set, the receiving device can rapidly switch its light transmission band to receive the fluorescence emitted by the sample after being excited in the light transmission band, and perform fluorescence imaging on the sample, meanwhile, hyperspectral imaging is realized by rapidly acquiring spectral information of fluorescence excited by each wave band.

Furthermore, fluorescence excited in the sample sequentially passes through the filtering device and the imaging lens and is converged on the surface of the area array photoelectric detector, the area array photoelectric detector converts optical signals into electric signals, and the image collector digitizes the electric signals and stores the electric signals in a computer.

Furthermore, a control module for controlling the wavelength of the light transmitted by the liquid crystal tunable filter is arranged on the liquid crystal tunable filter, the control module is connected to a computer, and the liquid crystal tunable filter can rapidly switch the light transmitting waveband to filter out stray light outside the light transmitting waveband.

Furthermore, the filter set is placed on the filter wheel, the filter wheel is connected with a motor, the motor is connected to a computer and controlled by the computer, and the filter set comprises a plurality of filters with different light transmission wave bands.

Furthermore, one end of the filter device, which is far away from the imaging lens, is connected with an exciter group filter used for filtering exciting light.

Further, the light source homogenizing module is arranged at one end of the exciter group for emitting light and enabling the light to be uniformly irradiated on the surface of the objective table.

Further, the exciter group comprises one or more lasers, the lasers are connected to a laser driving module, the laser driving module is connected to a computer, the wavelength of the excitation light of the exciter group is 400nm-1600nm, and the wavelength of the excitation light of the exciter group is 405nm, 488nm, 561nm, 640nm, 785nm, 808nm, 980nm or 1064 nm.

Further, the area array photodetector includes EMCCD, CCD, coms, scoms, InGaAs.

Further, the imaging lens comprises a fixed focus lens, a zoom lens, a body mirror and a microscope objective.

The utility model discloses a high spectrum live body fluorescence molecule imaging method, including following step:

s1: the single-wavelength exciter group uniformly irradiates a sample on the objective table through the light source homogenization module, and biomolecules or fluorescent probes in the sample emit fluorescence after being excited;

s2: the computer adjusts the wavelength transmitted by the liquid crystal tunable filter or rotates a filter wheel to perform two-dimensional space light splitting on the fluorescence, the fluorescence is divided into a plurality of narrow-band spectrums, the fluorescence is imaged on the area array photoelectric detector through an imaging lens, and continuous spectrum images or single-frame spectrum images are collected;

s3: the optical signal converged by the area array photoelectric detector through the imaging lens is converted into an electric signal by the photoelectric detector, and the electric signal is digitized by the image collector and then stored in a computer;

s4: and the computer performs data processing on the acquired series of spectral information and displays a processing result.

The utility model discloses a high spectrum live body fluorescence molecule imaging system can treat the biological process real-time observation under the test sample live body state, gathers continuous spectral image or gathers the spectral image of single frame through adjustable wavelength, carries out the accurate research of nature, ration, location in the molecular level.

Drawings

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

Fig. 1 is a schematic structural diagram of a hyperspectral in-vivo fluorescent molecular imaging system according to a preferred embodiment of the present invention.

Reference numerals: 1. an area array photodetector; 2. an imaging lens; 3. a light filtering means; 4. an exciter group optical filter; 5. a sample; 6. an object stage; 7. an exciter group; 8. a light source homogenizing module; 9. 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.

Example 1: as shown in fig. 1, the present invention discloses a hyperspectral living body fluorescence molecular imaging system, which comprises an object stage 6 for carrying a sample 5, wherein the object stage 6 is arranged in a darkroom box, and the object stage 6 is further provided with an illumination system and a life support system. A light source component and a receiving device are arranged above the objective table 6, the receiving device is positioned right above the objective table, and an included angle between a connecting line between the light source component and the sample 5 and the horizontal direction forms an acute angle. The light source assembly and the receiving device are also connected with a computer 9.

The light source component comprises an exciter group 7 and a light source homogenizing module 8, wherein the light source homogenizing module 8 is arranged at one end of the exciter group 7, which emits light, and enables the light to be uniformly irradiated on the objective table 6. The group of exciters 7 comprises one or more lasers, which are connected to a laser driver module, which is connected to a computer 9, the emission wavelength of the group of exciters 7 is rapidly switched by the computer 9 in the case of a plurality of lasers, and the switching of the lasers is controlled by the computer 9.

The receiving device comprises a filtering device, an imaging lens 2, an area array photoelectric detector 1 and an image collector which are sequentially arranged, an optical signal converged by the imaging lens 2 is converted into an electric signal by the area array photoelectric detector 1, and the electric signal is digitized and stored in a computer 9 by the image collector. The imaging lens 2 comprises a fixed focus lens, a zoom lens, a body mirror and a microscope objective. The area array photoelectric detector 1 comprises an EMCCD, a CCD, coms, scoms and InGaAs.

The light filtering device adopts a liquid crystal tunable filter 3 to split the fluorescence, a control module for controlling the wavelength of the transmitted light of the liquid crystal tunable filter 3 is arranged on the liquid crystal tunable filter, and the control module is connected to a computer 9. And one end of the liquid crystal tunable filter 3, which is far away from the imaging lens 2, is provided with an exciter group filter 4 for filtering exciting light. The Liquid Crystal Tunable Filter (LCTF) is designed based on the birefringence effect of liquid crystal molecules and the principle of polarized light interference. LCTF is a Lyot type tunable birefringent filter device, which is formed by cascading a plurality of Lyot components arranged in parallel, each stage comprises two mutually parallel polarizing plates, and the middle phase retarder consists of quartz plates with different thicknesses and tunable liquid crystal retarders. The natural light is changed into linearly polarized light after passing through the polarizing plate, when the linearly polarized light is in the quartz plate and the liquid crystal retarder, due to the double refraction effect of the quartz and the liquid crystal, extraordinary light (e light) which is flat and vibrates on the optical axis and ordinary light (o light) which vibrates vertical to the optical axis can be generated, the extraordinary light and the o light propagate in the same direction, but because the propagation speeds of the extraordinary light and the o light in the retarder are different, a certain optical path difference exists when the extraordinary light is emitted. Finally, when the P2 polarizer is transmitted, the vibration direction is the same, and therefore, an interference effect is generated, and a transmission curve similar to a sine wave is formed. And by applying voltages with different intensities to the two sides of the liquid crystal box, the liquid crystal molecular axis can deflect along with the direction of an electric field, the optical path difference of o light and e light is changed, namely the phase difference of the interference of the o light and the e light is changed, so that the transmittance curve can be quantitatively adjusted, and the waveform of the transmission curve is modulated. And finally, through cascade connection of multiple groups of Lyot pieces, the bandwidth of the transmission waveform can be further compressed, and finally, the selection of the transmission wave band can be realized.

The utility model discloses a high spectrum live body fluorescence molecule imaging system's imaging method, including following step:

s1: the exciter group 7 with single wavelength uniformly irradiates the sample 5 on the objective table 6 through the light source homogenization module 8, and biomolecules or fluorescent probes in the sample 5 emit fluorescence after being excited;

s2: the computer 9 adjusts the wavelength transmitted by the adjustable liquid crystal filter, performs two-dimensional space light splitting on the fluorescence, divides the fluorescence into a plurality of narrow-band spectrums, images the fluorescence on the area array photoelectric detector 1 through the imaging lens 2, and collects continuous spectrum images or single-frame spectrum images;

s3: the optical signal converged by the area array photoelectric detector 1 through the imaging lens 2 is converted into an electric signal by the photoelectric detector, and the electric signal is digitized by an image collector and then stored in a computer 9;

s4: the computer 9 performs data processing on the acquired series of spectrum cubes and displays the processing result.

The exciting light is uniformly irradiated on the sample 5, the emitted fluorescence is subjected to light splitting, and the fluorescence of the two-dimensional space of the sample 5 is recorded by a wavelength sequence. By analyzing the fluorescence spectrum cube data, localized, qualitative and quantitative analysis of the sample 5 can be performed. Compared with the traditional spectrum detection technology, the hyperspectral imaging technology has higher application value in the aspect of biomedical detection.

Example 2: a hyperspectral living body fluorescence molecule imaging system is different from embodiment 1 in that a filter set is adopted by a filter device, the filter set comprises a plurality of filters with different light transmission wave bands, the wave band difference of the filters is within +/-50 nm, the filter set is placed on a filter wheel, a motor is connected to the filter wheel, the motor is connected to a computer and controlled by the computer, two-dimensional or three-dimensional imaging of each spectrum can be rapidly acquired, and related purposes such as multispectral fluorescence separation and multi-information component splitting are achieved.

To sum up, the utility model discloses a high spectrum live body fluorescence molecule imaging system has following advantage:

1. the hyperspectral imaging technology is applied to in-vivo fluorescence imaging research, the imaging technology of high spatial resolution, high time resolution, high optical wavelength resolution, high sensitivity and high component analysis of the optical biological imaging technology is realized, the biological process in the in-vivo state is observed in real time, and the accurate research of qualitative, quantitative and positioning is carried out in the molecular level;

2. by adopting the liquid crystal tunable filter 3, the transmission waveband range of the liquid crystal tunable filter 3 is continuously adjustable, the switching speed is high, the spectrum scanning speed is high, the operation is simple, the gating wavelength band is narrow and wide, the spectrum resolution is high, no moving part is arranged, no mechanical jitter is generated, the aperture is large, the field angle is large, the volume is small, and the liquid crystal tunable filter has good optical characteristics;

3. separating overlapped fluorescence spectrum regions through an algorithm by adopting a hyperspectral imaging technology through a narrow-band optical filter, realizing fluorescence spectrum separation, and accurately and quantitatively analyzing a fluorescence marker;

4. an area array photoelectric detector 1 with a large target surface, high sensitivity, high QE and high photoelectric efficiency is used as an imaging original, and can be combined by one or more cameras to realize the fluorescence collection of 400-1700nm wide and high spectrum;

5. the adjustable wave band is wide and continuously adjustable, and the system is suitable for various biological fluorescent probes and nano materials. In addition, for multiple fluorescence labels in the sample 5, the exciter group 7 is rapidly switched, and the light transmission wave band of the liquid crystal tunable filter 3 is adjusted, so that the excited fluorescence is converged on the area array photoelectric detector 1 by the imaging lens 2 through the liquid crystal tunable filter 3, and a hyperspectral image of the sample 5 can be scanned.

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. A hyperspectral living body fluorescence molecule imaging system is characterized by comprising an object stage for bearing a sample, a computer for controlling the system and processing information, wherein the object stage is arranged in a darkroom box body, the object stage is also provided with an illuminating system and a life support system, a light source component for generating exciting light is arranged above the object stage, the light source component comprises one or more single-wavelength exciter groups and a light source homogenizing module, a receiving device for receiving fluorescence emitted by the sample after being excited is also arranged above the object stage, the receiving device comprises a filtering device, an imaging lens, an area array photoelectric detector and an image collector which are sequentially arranged, the filtering device is a liquid crystal tunable filter or a filtering sheet group, the receiving device can rapidly switch the light transmission waveband of the receiving device to receive the fluorescence emitted by the sample after being excited in the light transmission waveband, and carrying out fluorescence imaging on the sample, and simultaneously realizing hyperspectral imaging by rapidly acquiring spectral information of fluorescence excited by each wave band.

2. The hyperspectral in-vivo fluorescent molecular imaging system according to claim 1, wherein fluorescence excited in a sample sequentially passes through the filter device and the imaging lens and is converged on the surface of the area array photodetector, the area array photodetector converts optical signals into electrical signals, and the image collector digitizes the electrical signals and stores the electrical signals in a computer.

3. The hyperspectral in-vivo fluorescent molecular imaging system according to claim 1, wherein a control module for controlling the wavelength of light transmitted by the liquid crystal tunable filter is arranged on the liquid crystal tunable filter, the control module is connected to a computer, and the liquid crystal tunable filter can rapidly switch the light transmission band of the liquid crystal tunable filter to filter stray light outside the light transmission band.

4. The hyperspectral in-vivo fluorescent molecular imaging system of claim 1, wherein the filter set is placed on a filter wheel, the filter wheel is connected with a motor, the motor is connected with and controlled by a computer, and the filter set comprises a plurality of filters with different light transmission bands.

5. The hyperspectral in-vivo fluorescent molecular imaging system of claim 1, wherein one end of the filter device, which is away from the imaging lens, is connected with an exciter group filter for filtering exciting light.

6. The hyperspectral in vivo fluorescent molecular imaging system of claim 1, wherein the light source homogenizing module is disposed at one end of the light emitted by the exciter group and uniformly irradiates the surface of the stage.

7. The hyperspectral in-vivo fluorescent molecular imaging system of claim 1, wherein the exciter set comprises one or more lasers, the lasers are connected to a laser driving module, the laser driving module is connected to a computer, the wavelength of the excitation light of the exciter set is 400nm-1600nm, and the wavelength of the excitation light of the exciter set is 405nm, 488nm, 561nm, 640nm, 785nm, 808nm, 980nm or 1064 nm.

8. The hyperspectral in vivo fluorescent molecular imaging system of claim 1, wherein the area array photodetector comprises an EMCCD, a CCD, a coms, a scoms, an InGaAs.

9. The hyperspectral in vivo fluorescent molecular imaging system of claim 1, wherein the imaging lens comprises a fixed focus lens, a zoom lens, a stereoscope, and a microscope objective.