Multi-mode hyperspectral microscope imaging system and use method

Abstract

The invention relates to a multi-mode hyperspectral microscope imaging system and a use method, the multi-mode hyperspectral microscope imaging system comprises a multi-mode illumination assembly, a spectral imaging assembly and a control analysis assembly, the multi-mode illumination assembly comprises a high numerical aperture condenser, a second polarizer illumination channel, an illumination light source and a multi-path fluorescence excitation light source module; a light path of the illumination channel is composed of a turning reflector, a light path beam shrinking lens group and an illumination collimating lens, wherein the light path turning reflector, the illumination collimating lens and a stepping motor form a bright field and dark field switching module; the illumination light source is arranged on a coaxial light path of the illumination collimating lens; the spectral imaging assembly comprises a hyperspectral spectral imaging module arranged on a high-precision electric displacement table; the control analysis assembly comprises a work station and a controller. According to the invention, the accuracy of spectrum marking and sample analysis is improved; the system size is effectively reduced; continuity and integrity of spectrum acquisition can be realized; the experiment time is effectively shortened; and the applicability of the instrument is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of hyperspectral microscopy imaging, and particularly relates to a multimodal hyperspectral microscope imaging system and a usage method thereof. Background Art

[0002] The application of hyperspectral imaging technology mainly focuses on remote sensing, surface, vegetation, mineral detection, etc. Since this technology has the advantage of simultaneously obtaining image and spectral information of specific bands, when this technology is introduced into the biomedical field for tissue examination, it can be observed that different tissues or the same tissue due to lesions will cause changes in tissue component information in terms of content, structure and morphology, etc., and further cause changes in aspects such as waveform, peak intensity and wavelength of the spectral curve. This technology provides a strong basis for tissue identification. The technical field of this technical solution is hyperspectral microscopy imaging. Due to the flexibility of its system and the broadness of its application range, hyperspectral microscopes have shown great application potential in the fields of medical diagnosis and clinical research. With the continuous research and development of applications, there are more and more flexible transformations of general microscopes to meet the analytical requirements of the spectral dimension.

[0003] Domestically and internationally, there are relatively advanced types such as bright-field, dark-field, fluorescence hyperspectral microscopes, etc. In terms of implementation methods, a microscope generally has at most one or two imaging modes among bright-field, dark-field, and fluorescence. Since the application directions and scopes of the bright-field, dark-field, and fluorescence imaging modes are different, an experiment needs a hyperspectral imaging system that can meet different experiments and cover multiple directions.

[0004] The bright-field hyperspectral microscope uses illumination light to transmit a thin sample. The implementation structure is generally transmissive imaging, that is, the light source irradiates the sample and enters the imaging system through the sample. For example, the GaiaMicro series microscopic hyperspectral systems of Shuangli Heping in China have problems such as limited application range, strong background light interference, and light scattering and background noise that may affect spectral purity. It can only be applied to samples with relatively high spectral contrast, and it is impossible to accurately identify and label samples with relatively low spectral contrast, single cells, especially inside single cells.

[0005] The dark-field hyperspectral microscope generally uses a high numerical aperture optical system as a condenser on one side of the sample and blocks the light below the numerical aperture of the objective lens to achieve a dark-field effect. For example, the IMA hyperspectral dark-field microscope of Photonetc in Canada and the RTS2-Omn i-Imager dark-field scattering microscopic hyperspectral spectrometer of Beijing Zhuoli HanGuang have problems: this implementation method significantly reduces the light utilization rate of the illumination light source, and to obtain spectral imaging signals, it is necessary to increase the power of the illumination light source or extend the camera exposure time.

[0006] Fluorescence hyperspectral microscopes are generally designed mainly based on the coaxial fluorescence excitation principle of fluorescence microscopes. For example, the fluorescence hyperspectral microscope of Shuanglihepu in China has the problem that due to the influence of the optical element dichroic mirror, the finally obtained spectral range is only a part of the sample fluorescence spectrum, the spectral range is narrow, especially the fluorescence spectrum is not continuous. In general, only the fluorescence signal of one channel can be detected at a time, and the detection of fluorescence signals of multiple channels requires multiple detections.

[0007] In the document with the application number "201911411930.5", "A microbial multimodal imaging system and a microbial multimodal imaging detection method" are disclosed. The system includes a laser group, a fiber optic beam splitter suitable for splitting the incident first imaging laser beam into a reference beam and a sample beam or passing through the incident second imaging laser beam, and an illumination unit suitable for generating a corresponding first structured light according to the incident sample beam and the loaded first simulated holographic image and irradiating the first structured light onto the sample or generating a second structured light according to the incident second imaging laser beam and the loaded second simulated holographic image and irradiating the second structured light onto the sample. The existing problem is that this patent uses the same laser light source to irradiate the sample through the reference optical path and the diffraction optical path respectively and image separately, realizing label-free three-dimensional refractive index images and three-dimensional fluorescence imaging images. In implementation, two coherent illumination light sources are required and are respectively integrated in two imaging systems. It is a method to improve the spatial and temporal resolution of fluorescence imaging, and this scheme cannot be applied in the field of hyperspectral microscopy imaging. Summary of the Invention

[0008] The present invention relates to a multimodal hyperspectral microscope imaging system and a usage method, in order to overcome the problems existing in the prior art, such as the single application direction of hyperspectral imaging, the low utilization rate of dark field hyperspectral illumination light sources, the narrow spectral range of fluorescence hyperspectral detection, and the inability to simultaneously obtain the spectra of multiple fluorescence channels.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows: A multimodal hyperspectral microscope imaging system includes a multimodal illumination component, a spectral imaging component, and a control and analysis component;

[0010] The multimodal illumination component includes a high numerical aperture condenser lens, a second polarizer illumination channel, an illumination light source, and a multi-channel fluorescence excitation light source module sequentially arranged on the light path of the spectral imaging component; the light path of the illumination channel is composed of a light path turning mirror, a light path beam shrinking lens group, and an illumination collimating lens, wherein the light path turning mirror and the illumination collimating lens are respectively connected to a stepping motor and jointly form a bright field and dark field switching module; the illumination light source is arranged on the coaxial light path of the illumination collimating lens;

[0011] The spectral imaging component includes a hyperspectral splitting and imaging module arranged on a high-precision electric displacement stage;

[0012] The control and analysis component includes a workstation and a controller.

[0013] Furthermore, the above hyperspectral spectroscopic imaging module includes a main camera, a first polarizer, a spectral spectroscopic module, an imaging objective lens, and a sub-camera.

[0014] Furthermore, a method for using the above multi-modal hyperspectral microscope imaging system includes the following steps:

[0015] Step 1: Turn on the multi-modal hyperspectral microscope imaging system with bright field, dark field, and fluorescence capabilities.

[0016] Step 2: Place the biological sample on the high-precision electric displacement stage.

[0017] Step 3: Control the high-precision electric displacement stage, and observe and select the sample area position to be spectrally scanned and photographed through the sub-camera.

[0018] Step 4: Confirm the selected shooting mode.

[0019] Step 5: The hyperspectral spectroscopic imaging module transmits the spectral data obtained in different modalities to the workstation for data processing and analysis.

[0020] Furthermore, the shooting modes in the above Step 4 include:

[0021] a. Default bright field mode, controlling the illumination light source (6) through the controller (9) for illuminating and photographing the biological sample.

[0022] b. Controlling the bright field and dark field switching module, adjusting the illumination of the illumination light source (6) to the dark field mode, adjusting the first polarizer (1.2) and the second polarizer (4) to remove stray interference light, and then taking a photograph.

[0023] c. Controlling the bright field and dark field switching module to move to a non-working area, starting the multi-channel fluorescence excitation light source (7) to work, adjusting the illumination angle for fluorescence photography to obtain spectral data.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] 1. This solution integrates bright field, dark field, and fluorescence (continuous spectrum), and designs a system that meets various hyperspectral microscopic imaging experimental modes. The three hyperspectral imaging modes of bright field, dark field, and fluorescence can work independently, or can analyze a sample from different hyperspectral imaging angles, corroborating each other, and improving the accuracy of spectral labeling and sample analysis.

[0026] 2. In the system of the present invention, the bright field, dark field, and fluorescence modes share one illumination channel. Meanwhile, the bright field and dark field share one illumination light source, and different spectral modes are multiplexed, effectively reducing the system volume.

[0027] 3. The fluorescence illumination part is independent of the hyperspectral imaging module. Its implementation method is close to Ti rf (total internal reflection fluorescence), without blocking the light source, effectively improving the light source utilization rate. At the same time, all fluorescence spectra are obtained without loss in the full band, realizing the continuity and integrity of spectral acquisition.

[0028] 4. Since the present invention cancels coaxial fluorescence excitation and does not use a fluorescence turntable and dichroic mirror, etc., it can realize simultaneous imaging of multiple fluorescence, shorten the experimental time, and help users reduce the cost of experimental samples.

[0029] 5. In the method of the present invention, in step four, according to different biological sample conditions and experimental objectives, the stepper motors in the two displacement modules can be adjusted to switch among the bright field, dark field, and fluorescence hyperspectral modes of hyperspectral imaging, and find a suitable spectral experimental mode. The operation method is simple. In the fluorescence hyperspectral mode, the stepper motor in the multi-channel fluorescence excitation light source is controlled to adjust the light source to find a suitable illumination angle for fluorescence excitation. This method can adapt to different biological sample containers and samples with different thicknesses, improving the applicability of the instrument. Adjusting the two polarizing plates can improve the signal-to-noise ratio and optimize the acquired image quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic structural diagram of a multi-modal hyperspectral microscope imaging system;

[0031] Figure 2 Schematic diagram of the illumination module in bright field mode;

[0032] Figure 3 Schematic diagram of the illumination module in dark field mode;

[0033] Figure 4 Schematic diagram of the illumination module in fluorescence mode;

[0034] Wherein the reference numerals are as follows: 1 - hyperspectral spectroscopic imaging module, 1.1 - main camera, 1.2 - first polarizing plate, 1.3 - imaging objective lens, 1.4 - sub-camera, 1.5 - spectral spectroscopic module, 2 - high-precision electric displacement stage, 3 - high numerical aperture condenser, 4 - second polarizing plate, 5 - illumination channel, 5.1 - optical path turning mirror, 5.2 - optical path beam reducing lens group, 5.3 - illumination collimating lens, 6 - illumination light source, 7 - multi-channel fluorescence excitation light source, 8 - workstation, 9 - controller. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present invention will be further described in detail below with reference to the drawings and embodiments.

[0036] See Figures 1-4 Figures 1-4 , a multimodal hyperspectral microscope imaging system, including a multimodal illumination component, a spectral imaging component and a control and analysis component.

[0037] The multimodal illumination component includes a high numerical aperture condenser lens 3, a second polarizer 4, an illumination channel 5, an illumination light source 6 and a multi-channel fluorescence excitation light source 7, which are sequentially arranged on the outgoing light path of the spectral imaging component; the optical path of the illumination channel 5 is composed of an optical path turning mirror 5.1, an optical path beam shrinking lens group 5.2 and an illumination collimating lens 5.3, wherein the optical path turning mirror 5.1 and the illumination collimating lens 5.3 are respectively connected to a stepping motor and jointly form a bright field and dark field switching module; the illumination light source 6 is arranged on the coaxial light path of the illumination collimating lens 5.3; the spectral imaging component includes a hyperspectral spectroscopic imaging module 1 arranged on a high-precision electric displacement stage 2, and the hyperspectral spectroscopic imaging module 1 includes a main camera 1.1, a first polarizer 1.2, a spectral spectroscopic module 1.5, an imaging objective lens 1.3 and a sub-camera 1.4; the control and analysis component includes a workstation 8 and a controller 9.

[0038] The high numerical aperture condenser lens 3, in cooperation with the bright field and dark field switching module and the multi-channel fluorescence excitation light source 7, realizes the switching of different modes of bright field, dark field and fluorescence; the second polarizer 4 is used in cooperation with the first polarizer 1.2; the illumination channel 5 is a common channel for bright field, dark field and fluorescence illumination, and is switched through the bright field and dark field switching module, and the switching method is realized by the movement of the stepping motor; the illumination light source 6 is a common illumination light source for bright field and dark field, and the switch and brightness adjustment are controlled by the controller 9 system; the multi-channel fluorescence excitation light source 7 has a displacement module stepping motor switching function and is composed of a multi-channel LED light source module, and can select common fluorescence excitation bands as needed, such as 405nm, 488nm, 543nm, 561nm, 656nm, 750nm, etc.

[0039] The usage method of the multimodal hyperspectral microscope imaging system includes the following steps:

[0040] Step 1: Turn on the multimodal hyperspectral microscope imaging system with bright field, dark field and fluorescence functions;

[0041] Step 2: Place the biological sample on the high-precision electric displacement stage 2;

[0042] Step 3: Control the high-precision electric displacement stage 2, and observe and select the sample area position to be spectrally scanned and photographed through the sub-camera 1.4;

[0043] Step 4: Confirm the selected shooting mode:

[0044] a. The default bright-field mode, where the illumination light source 6 is controlled by the controller 9 to illuminate and photograph the biological sample.

[0045] b. Control the bright-field / dark-field switching module to adjust the illumination of the illumination light source 6 to the dark-field mode, and adjust the first polarizer 1.2 and the second polarizer 4 to remove stray interference light for photographing.

[0046] c. Control the bright-field / dark-field switching module to move to the non-working area, and the multi-channel fluorescence excitation light source 7 starts to work. Control the stepper motor in the multi-channel fluorescence excitation light source 7 to find a suitable fluorescence adjustment illumination angle for fluorescence photographing; during the photographing process, adjust the controller 9 to realize the excitation of the sample fluorescence by different wavelength light source combinations to obtain spectral data.

[0047] Step Five: The hyperspectral spectroscopic imaging module 1 transmits the spectral data obtained in different modalities selected in Step Four to the workstation 8 for data processing and analysis.

[0048] The workstation 8 acquires all the data collected by the hyperspectral spectroscopic imaging module 1 and sends it to the controller 9 system through software instructions to realize the control of the high-precision electric displacement stage 2, the bright-field / dark-field switching module, the stepper motor in the multi-channel fluorescence excitation light source 7, and all the light source switches and brightness of the illumination light source 6 and the multi-channel fluorescence excitation light source 7.

[0049] The above is the description of the specific implementation of the present invention, rather than the limitation of the present invention. Those skilled in the relevant technical field can also make various equivalent technical solutions without departing from the scope of the present invention. Therefore, all equivalent technical solutions should be included in the patent protection scope of the present invention.

Claims

1. A multimodal hyperspectral microscope imaging system, characterized in that: It includes a multimodal lighting component, a spectral imaging component, and a control and analysis component; The multimodal lighting component includes a high numerical aperture condenser lens (3), a second polarizer (4), an illumination channel (5), an illumination light source (6), and a multi-channel fluorescence excitation light source module (7) sequentially arranged on the light path emerging from the spectral imaging component; the light path of the illumination channel (5) is composed of a light path turning mirror (5.1), a light path beam shrinking lens group (5.2), and an illumination collimating lens (5.3), wherein the light path turning mirror (5.1) and the illumination collimating lens (5.3) are respectively connected to a stepping motor to jointly form a bright field and dark field switching module; the illumination light source (6) is arranged on the coaxial light path of the illumination collimating lens (5.3); The spectral imaging component includes a hyperspectral spectroscopic imaging module (1) arranged on a high-precision electric displacement stage (2); The control and analysis component includes a workstation (8) and a controller (9).

2. The multimodal hyperspectral microscope imaging system according to claim 1, wherein: The hyperspectral spectroscopic imaging module (1) includes a main camera (1.1), a first polarizer (1.2), a spectral spectroscopic module (1.5), an imaging objective lens (1.3), and a sub-camera (1.4).

3. The method for using a multimodal hyperspectral microscope imaging system according to claim 1, wherein: It includes the following steps: Step 1: Turn on the multimodal hyperspectral microscope imaging system with bright field, dark field, and fluorescence; Step 2: Place the biological sample on the high-precision electric displacement stage (2); Step 3: Control the high-precision electric displacement stage (2), and observe and select the sample area position to be spectrally scanned and photographed through the sub-camera (1.4); Step 4: Confirm the selected shooting mode; Step 5: The hyperspectral spectroscopic imaging module (1) transmits the spectral data obtained in different modes to the workstation (8) for data processing and analysis.

4. The method for using a multimodal hyperspectral microscope imaging system according to claim 3, wherein: The shooting modes in the said Step 4 include a. The default bright field mode, where the illumination light source (6) is controlled by the controller (9) to illuminate and photograph the biological sample; b. Control the bright field and dark field switching module to adjust the illumination of the illumination light source (6) to the dark field mode, and adjust the first polarizer (1.2) and the second polarizer (4) to remove stray interference light for shooting; c. Control the bright field and dark field switching module to move to a non-working area, the multi-channel fluorescence excitation light source (7) starts to work, and adjust the illumination angle to perform fluorescence shooting to obtain spectral data.

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