CN215339511U - Scanning type microscopic hyperspectral imaging system - Google Patents
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Abstract
本实用新型适用于显微成像技术领域，提供了一种扫描式显微高光谱成像系统，包括：激发光源；样本显微组件，包括放置待测样本的样品台以及对待测样本进行显微的物镜组；振镜扫描组件，产生不同偏转角度的激发光，不同偏转角度的激发光经物镜组聚焦到待测样本上，以对待测样本进行扫描；滤光部件，设置于所述激发光出射光路和反射光路上，对入射激光和反射荧光进行滤波；图像探测模块，设置于反射路径上，用于接收滤波后的混合荧光并进行显微高光谱成像。本实用新型利用光的点扫描成像方式，提高了系统的空间分辨率，结合物镜组不同的放大倍率，可实现对多种目标的高分辨率探测，还消除了样品机械运动对成像造成的模糊和伪影。
The utility model is suitable for the field of microscopic imaging technology, and provides a scanning type microscopic hyperspectral imaging system, comprising: an excitation light source; The objective lens group; the galvanometer scanning component generates excitation light with different deflection angles, and the excitation light with different deflection angles is focused on the sample to be tested by the objective lens group, so as to scan the sample to be tested; the filter part is arranged on the output of the excitation light The incident laser light and the reflected fluorescence are filtered on the emission light path and the reflection light path; the image detection module is arranged on the reflection path and is used for receiving the filtered mixed fluorescence and performing microscopic hyperspectral imaging. The utility model utilizes the point scanning imaging method of light to improve the spatial resolution of the system, and combined with the different magnifications of the objective lens group, high-resolution detection of various targets can be realized, and the blur caused by the mechanical movement of the sample to the imaging can be eliminated. and artifacts.
Description
Technical Field
The utility model belongs to the technical field of microscopic imaging, and particularly relates to a scanning type microscopic hyperspectral imaging system.
Background
The hyperspectral imaging is taken as a branch of spectral imaging technology, is emerging since the 80 th generation of the 20 th century, and is widely applied to emerging technologies in the fields of agriculture, mineralogy, aerology, life science and the like. Compared with the traditional imaging technology, the method can obtain target image information and spectral information of specific wave bands, the spectral resolution of hyperspectral imaging can reach below 10nm, and the method can instantaneously record the spectral information of dozens or even hundreds of continuous narrow wave bands of each pixel in a field angle. In recent years, with the penetration of microscopy to various aspects of biomedical detection, many reports of combining hyperspectral technology with microscopy imaging have appeared, such as: chromosome recognition, cancer diagnosis, skin disease examination, nondestructive testing of food, agricultural products and the like, cell function research and the like.
The existing cell detection technology is difficult to obtain spatial information and composition information of a sample at the same time, and the microscopic hyperspectral technology can well present appearance information and internal chemical composition information of cells, so that the research of a microscopic hyperspectral imaging system has great significance to the cells and even the whole life science field. In the field of biomedical research, autofluorescence detection of biological tissues has been widely applied to prevention and diagnosis research of difficult and complicated diseases, while hyperspectral microimaging technology combined with microscopy and spectral imaging technology can be used for quantitative analysis of pathology, and compared with the traditional medical imaging method, the hyperspectral microimaging method can provide richer spectral component information and objective diagnosis standards, and has a wide application prospect in the field of biomedical.
Most of the existing hyperspectral imaging systems mostly adopt a push-and-sweep scanning mode and a staring scanning mode, but autofluorescence signals of most organisms are weak, and if the push-and-sweep scanning mode and the staring scanning mode are used for imaging, the influence of other background fluorescence is easily caused, so that data acquisition is inaccurate, the resolution of a spectrum and a reconstructed image is low, and meanwhile, motion artifacts are easily caused for a liquid sample or a living sample. Meanwhile, the existing hyperspectral imaging system cannot simultaneously obtain a spectrum and a microscopic image through one-time scanning, and cannot comprehensively analyze a sample through integrated map information.
SUMMERY OF THE UTILITY MODEL
The embodiment of the utility model provides a scanning type microscopic hyperspectral imaging system, and aims to solve the technical problem of low resolution of the conventional microscopic hyperspectral imaging.
The embodiment of the utility model is realized in such a way that a scanning type microscopic hyperspectral imaging system comprises:
the excitation light source is used for providing excitation light for irradiating a sample to be detected;
the sample microscopic assembly comprises a sample table for placing the sample to be detected and an objective lens group for carrying out microscopy on the sample to be detected;
the galvanometer scanning component comprises a scanning galvanometer, a scanning lens and a sleeve lens which are sequentially arranged along an emergent light path of the exciting light, the exciting light generates exciting light with different deflection angles after being adjusted by the scanning galvanometer and the scanning lens, the exciting light with different deflection angles enters the objective lens group after being collimated by the sleeve lens and is focused on a sample to be detected so as to scan the sample to be detected, the sample to be detected emits fluorescence after being scanned and excited by the exciting light, the fluorescence and the exciting light reflected by the sample to be detected are mixed to form mixed fluorescence, and the mixed fluorescence is conducted along a reflection light path;
the light filtering component is arranged on the exciting light emergent light path and the reflecting light path and is used for filtering incident laser and reflected fluorescence;
the image detection module is arranged on the reflection path and used for receiving the mixed fluorescence filtered by the filtering component and performing microscopic hyperspectral imaging;
wherein the system further comprises:
the plane reflector is arranged on an emergent light path of the exciting light and is positioned between the sleeve lens and the objective lens group, and the exciting light collimated by the sleeve lens enters the objective lens group after being reflected by the plane reflector.
Preferably, the image detection module includes:
the light path switching component is arranged on the reflection path and used for receiving the mixed fluorescence filtered by the filtering component and enabling the filtered mixed fluorescence to be conducted along a first conduction light path or a second conduction light path;
the spectrometer is arranged on the first conduction light path and used for receiving the fluorescence conducted along the first conduction light path and separating the mixed fluorescence to form a spectrum of the sample to be detected;
and the imaging device is arranged on the second conduction light path and is used for receiving the fluorescence conducted along the second conduction light path so as to form a microscopic image of the sample to be detected.
Preferably, the first light transmission path is provided with a fluorescence color filter and a first focusing lens, and the fluorescence transmitted along the first light transmission path sequentially passes through the fluorescence color filter and the first focusing lens and then is emitted to the spectrometer;
and the fluorescence transmitted along the second transmission light path is emitted to the imaging device after passing through the second focusing lens.
Preferably, the objective lens group is inverted below the sample stage.
Preferably, the sample microscope assembly further comprises a halogen lamp, a phase difference ring and a condenser lens which are arranged above the sample stage and are sequentially close to the sample stage.
Preferably, the scanning microscopic hyperspectral imaging system further comprises:
and the space beam expanding filter is arranged on the emergent light path of the exciting light, is positioned between the exciting light source and the filtering component and is used for expanding the exciting light and eliminating stray light.
Preferably, the spatial beam expanding filter comprises a first beam expanding lens, a pinhole, a second beam expanding lens and an excitation color filter which are sequentially arranged along an emergent light path of the excitation light.
Preferably, the filtering component is a dichroic mirror.
Preferably, the optical path switching component is an adjustable plane mirror.
Preferably, the excitation light source is a multi-thread laser capable of emitting excitation light of multiple wavelengths.
The utility model achieves the following beneficial effects: the exciting light is adjusted by arranging the galvanometer scanning component, the exciting light with different deflection angles is generated to scan a sample to be detected, so that the spatial resolution of the system is improved by utilizing a point scanning imaging mode of light, the high-resolution detection of various targets can be realized by combining different magnification ratios of the lens group, and the blurring and the artifacts caused by the mechanical movement of the sample to the imaging are also eliminated.
Drawings
FIG. 1 is a schematic structural diagram of a scanning microscopic hyperspectral imaging system in one embodiment of the utility model;
fig. 2 is a three-dimensional structural diagram of a scanning microscopic hyperspectral imaging system in one embodiment of the utility model.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the utility model and are not intended to limit the utility model.
Example one
Referring to fig. 1-2, a scanning microscopic hyperspectral imaging system in accordance with a first embodiment of the utility model is shown, which includes an excitation light source 1, a galvanometer scanning component 30, a sample microscopic component 40, a filter component 5, and an image detection module 50. Wherein:
the excitation light source 1 is used for providing excitation light for irradiating a sample to be detected, the excitation light emits to the sample to be detected along a set emergent light path, the sample to be detected can generate a fluorescence signal after being irradiated and excited by the excitation light, and the fluorescence signal can be used for subsequent hyperspectral imaging. Preferably, the excitation light source 1 may be a multi-thread laser capable of emitting excitation light with multiple wavelengths, so that excitation of fluorescence signals in multiple visible light ranges can be satisfied, and the universality of the system is improved. By way of example and not limitation, the multi-thread laser may specifically emit excitation lights of 405nm, 488nm and 561nm, but is not limited thereto, and the number of types of the excitation lights may be increased or decreased according to actual needs, or excitation lights with different wavelengths may be adopted.
The sample microscope assembly 40 includes a sample stage 12 on which a sample to be measured is placed and an objective lens group 10 for microscopic examination of the sample to be measured. In the present embodiment, the objective lens group 10 is inverted below the sample stage 12, and this inverted microstructure is favorable for imaging semitransparent or even transparent samples, and is further favorable for realizing transmission and fluorescence imaging of cells and the like. In some cases of the present embodiment, the sample microscope assembly 40 further includes a halogen lamp 16, a phase difference ring 15, and a condenser 14 disposed above the sample stage 12 and disposed in sequence close to the sample stage 12, and by mounting the halogen lamp 16 and the like, bright field images can be acquired at the same time as fluorescent imaging, and different spatial resolutions can be obtained by switching the objective lens group 10.
The galvanometer scanning component 30 is arranged on an emergent light path of the exciting light and used for receiving the exciting light and adjusting the exciting light to generate the exciting light with different deflection angles, and the exciting light with different deflection angles is focused on a sample to be measured through the objective lens group 10 to scan the sample to be measured, so that the hyperspectral imaging is performed in a point scanning imaging mode of light. Specifically, in the present embodiment, the galvanometer scanning assembly 30 specifically includes a scanning galvanometer 6, a scanning lens 7, and a sleeve lens 8 sequentially arranged along an exit light path of the excitation light, and the excitation light enters the objective lens group 10 after being adjusted by the scanning galvanometer 6 and the scanning lens 7 and then collimated by the sleeve lens 8. In addition, in some optional cases of this embodiment, the system may further include a plane mirror 9, where the plane mirror 9 is disposed on an exit light path of the excitation light and located between the sleeve lens 8 and the objective lens group 10, and the excitation light collimated by the sleeve lens 8 enters the objective lens group 10 after being reflected by the plane mirror 9. By arranging the plane mirror 9 to switch the horizontally conducted excitation light into vertically conducted excitation light, the galvanometer scanning assembly 30 and the sample microscopic assembly 40 can be arranged horizontally and vertically one by one, which is beneficial to reducing the volume of the system. Wherein the scanning galvanometer 6 can be Thorlabs GVS212, the wavelength range is 400-.
It should be noted that, the sample to be measured emits fluorescence after being scanned and excited by the excitation light, and the sample to be measured also reflects the excitation light, and the excitation light reflected by the sample to be measured and the fluorescence emitted by the sample to be measured are mixed together to form mixed fluorescence, and the mixed fluorescence is conducted along the reflection light path.
The filtering component 5 is disposed on the reflection path and is configured to receive the reflected mixed fluorescence and filter fluorescence in a non-interesting waveband, and the filtered mixed fluorescence continues to be transmitted along the reflection light path. Meanwhile, the filter component 5 is also arranged on an emergent light path of the exciting light, and the exciting light enters the galvanometer scanning component 30 after being reflected by the filter component 5, so that an optical device can be saved in the system, the cost is reduced, and the system can be integrated more highly.
The image detection module 50 is disposed on the reflection path, and is configured to receive the mixed fluorescence filtered by the filtering component 5 and perform hyperspectral microscopy imaging. Specifically, the image detection module 50 specifically includes an optical path switching component 17, a spectrometer 22, and an imaging device 19, where the optical path switching component 17 is disposed on the reflection path, and is configured to receive the mixed fluorescence filtered by the filtering component 5 and enable the filtered mixed fluorescence to be conducted along the first conduction optical path or the second conduction optical path. The spectrometer 22 is disposed on the first light transmission path and configured to receive the fluorescence transmitted along the first light transmission path and separate the mixed fluorescence (i.e., separate the fluorescence signal from the mixed fluorescence) to form a spectrum of the sample to be measured. The imaging device 19 is disposed on the second conduction optical path and is configured to receive the fluorescence propagating along the second conduction optical path to form a microscopic image of the sample to be measured. In addition, the first light transmission path is further provided with a fluorescence color filter 20 and a first focusing lens 21, fluorescence transmitted along the first light transmission path sequentially passes through the fluorescence color filter and the first focusing lens and then is emitted to the spectrometer, the fluorescence color filter 20 is used for filtering stray light in the fluorescence signal, and after filtering, the fluorescence signal is focused by the first focusing lens 21 and then enters the spectrometer 22. The spectrometer 22 selects Andor Kymera 328i-B1, the built-in grating has three specifications of 150g/mm, 600g/mm and 1200g/mm, the theoretical spectral resolution can reach 0.62nm, 0.15nm and 0.07nm respectively, but for the spectral resolution, the size of an incident slit can also influence the spectral resolution. A 150g/mm grating with a spectral resolution of 1.56nm was used with a slit of 150um, depending on the desired study sample requirements. Similarly, a second focusing lens 18 is further disposed on the second conduction optical path, and the fluorescence guided along the second conduction optical path passes through the second focusing lens and then is emitted to the imaging device, that is, the fluorescence signal is focused by the second focusing lens 18 and then enters the imaging device 19.
By way of example and not limitation, in the present embodiment, the filtering component 5 may be specifically a dichroic mirror, and the optical path switching component 17 may be specifically an adjustable plane mirror 9. The imaging device 19 may specifically be a CCD or an smcmos camera. In order to realize sampling at a higher speed, the imaging device 19 selects Andor zyla5.5sCMOs, the effective pixel number is 2560 x 2160, the pixel size is 6.5um x 6.5um, the full frame rate is 40fps, the pixel clock is 200MHz/560MHz, only 2000 x 30 pixel areas are selected according to the required acquisition requirement, and the sampling frequency is 500Hz under the condition of exposure for 1.4 ms.
It should be understood that, since the light is reversible, the reflected light path of the mixed fluorescence will be reciprocal to the emitted light path of the excitation light, i.e., the mixed fluorescence will return.
Further, in some cases of this embodiment, the scanning type microscopic hyperspectral imaging system further includes a spatial beam expanding filter 60, where the spatial beam expanding filter 60 is disposed on the emergent light path of the excitation light, and is located between the excitation light source 1 and the filtering component 5, and is used to expand the excitation light and eliminate the stray light. Specifically, in the present embodiment, the spatial beam expanding filter 60 specifically includes the first beam expanding lens 2, the pinhole, the second beam expanding lens 3, and the excitation color filter 4, which are arranged in this order along the exit optical path of the excitation light. The laser spot can be enlarged by 40 times through the spatial beam expanding filter 60, incident light is more uniform, and the incident spot is enlarged as much as possible, so that a focused spot coming out of the objective lens can be as small as possible, and the spatial resolution can be improved. For example, in the case of a 40X objective, the spot diameter is only 0.41 um.
Based on the above structure, please refer to fig. 2, the working principle of the scanning type microscopic hyperspectral imaging system is as follows: an excitation light source 1 emits excitation light, the excitation light sequentially passes through a first beam expanding lens 2, a pinhole, a second beam expanding lens 3 and an excitation color filter 4, then enters a scanning vibrating mirror 6 after being reflected by a light filtering part 5, is adjusted in the scanning vibrating mirror 6 to form excitation light with different deflection angles, and then is focused on the same focal plane by a scanning lens 7, and is collimated by a sleeve lens 8 and then enters an objective lens group 10 after being reflected by a plane reflecting mirror 9, so that a sample to be detected on a sample stage 12 is scanned;
the original path of the mixed fluorescence generated by scanning returns, after the mixed fluorescence reaches the light filtering component 5, the mixed fluorescence is transmitted to the light path switching component 17 through the light filtering component 5, and the fluorescence signal is transmitted to the imaging device 19 and the spectrometer 22 in sequence through the switching of the light path switching component 17, so that the spectrum and the microscopic image can be obtained simultaneously by one-time scanning.
In addition, the scanning microscopic hyperspectral imaging system further comprises a computer 14, wherein the computer 14 is respectively electrically connected with the optical path switching component 17, the spectrometer 22, the imaging device 19 and the scanning galvanometer 6 and is used for synchronously controlling the optical path switching component 17, the spectrometer 22, the imaging device 19 and the scanning galvanometer 6. Specifically, the computer 14 is connected to the scanning galvanometer 6 through the NI data acquisition card 11, and each time the scanning galvanometer 6 scans a point, a signal for triggering the acquisition of a spectrum is sent to the NI data acquisition card 11, so that the computer 14 synchronously controls the optical path switching component 17, the spectrometer 22 and the imaging device 19 to perform spectrum imaging.
In summary, the scanning microscopic hyperspectral imaging system in the embodiment at least has the following beneficial effects:
1. the exciting light is adjusted by arranging the galvanometer scanning component 30, the exciting light with different deflection angles is generated to scan a sample to be detected, the spatial resolution of the system is improved by utilizing a point scanning imaging mode of light, the high-resolution detection of various targets can be realized by combining different magnification ratios of the lens group 10, and the blurring and the artifacts caused by the mechanical movement of the sample to the imaging are eliminated;
2. the dichroic mirror is adopted for filtering, and then the grating in the spectrometer is adopted for splitting the spectrum, namely the grating is adopted for splitting the spectrum, the spectral resolution can reach 1nm, and the liquid crystal tunable filter and the acousto-optic tunable filter have higher spectral resolution, so that different fluorescence emission peaks can be better distinguished, and crosstalk is avoided;
3. the whole galvanometer scanning assembly 30 is designed into a whole module (as shown in figure 2), and can be conveniently carried in various microscopes, so that the modularization and simplification of the construction of a hyperspectral microscope system are realized;
4. through the setting of the parameters, the main performance index of the system is high, and the method specifically comprises the following steps: spectral range: 500-750nm, spectral resolution: <2nm, band number: 1000. spatial resolution: depending on the magnification of the objective lens, the magnification is 0.41um at 40 times.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the utility model. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the utility model, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986204A (en) * | 2021-04-16 | 2021-06-18 | 深圳技术大学 | Scanning type microscopic hyperspectral imaging system |
| CN116735562A (en) * | 2023-08-15 | 2023-09-12 | 深圳湾实验室 | Three-dimensional dynamic microscopic imaging system, method and storage medium |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986204A (en) * | 2021-04-16 | 2021-06-18 | 深圳技术大学 | Scanning type microscopic hyperspectral imaging system |
| CN116735562A (en) * | 2023-08-15 | 2023-09-12 | 深圳湾实验室 | Three-dimensional dynamic microscopic imaging system, method and storage medium |
| CN116735562B (en) * | 2023-08-15 | 2024-07-16 | 深圳湾实验室 | Three-dimensional dynamic microscopic imaging system, method and storage medium |
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