Light sheet microscopic system based on spatial light modulator

Abstract

The invention belongs to the technical field of microscopic imaging, and discloses a light sheet microscopic system based on a spatial light modulator, comprising: an LED light source; the light beam shaping unit is used for shaping light rays emitted by the LED light source into uniform and collimated illumination light beams and irradiating the illumination light beams to the spatial light modulator through the projection prism; the projection imaging unit is used for shaping the light field modulated by the spatial light modulator into a light sheet meeting requirements and irradiating the light sheet to a target object; the camera is used for acquiring an image of the target object under the illumination of the light sheet; and the processing unit is used for encoding the spatial light modulator and analyzing and synthesizing the shot image to obtain a three-dimensional light sheet image. The position of the light sheet can be accurately controlled, and the light path is simple in structure and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of microscopic imaging, and in particular relates to a light sheet microscopic system based on a spatial light modulator. Background Art

[0002] Light sheet microscopy is a microscopic imaging technique that uses a lateral light sheet to illuminate the sample and collect images perpendicular to the light sheet direction. It has been widely used in the biomedical field due to its fast speed, low phototoxicity and suitability for three-dimensional imaging of large samples.

[0003] In the process of realizing the present invention, the inventors found that there are at least the following problems in the prior art: in the existing light sheet microscopy imaging system, a laser light source and a laser shaping system are used to form light sheet illumination, and the optical path structure is complex and the cost is high; in the existing light sheet microscopy imaging system, due to the use of a laser light source, the imaging quality is affected by laser speckle and temperature drift; the layer-by-layer scanning imaging within the depth of field is affected by the step scanning speed of the piezoelectric ceramic drive stage, and the speed is slow and the efficiency is low.

[0004] The above description of the background technology is only for facilitating an in-depth understanding of the technical solution of the present invention (such as the technical means used, the technical problems solved and the technical effects produced), and should not be regarded as an admission or any form of implication that the information constitutes prior art already known to those skilled in the art. Summary of the invention

[0005] The present invention aims to solve the above technical problems at least to a certain extent. To this end, the present invention aims to provide a light sheet microscopy system based on a spatial light modulator.

[0006] The technical solution adopted by the present invention is:

[0007] A light sheet microscopy system based on a spatial light modulator, comprising:

[0008] LED light source;

[0009] A beam shaping unit, used to shape the light emitted by the LED light source into a uniform and collimated illumination beam, and irradiate it onto the spatial light modulator through a projection prism;

[0010] A projection imaging unit is used to shape the light field modulated by the spatial light modulator into a light sheet that meets the requirements and irradiate it onto the target object;

[0011] A camera collects images of the target object under the illumination of the light sheet;

[0012] The processing unit is used to encode the spatial light modulator and to analyze and synthesize the captured images to obtain three-dimensional light sheet imaging.

[0013] Preferably, the processing unit analyzes and synthesizes the captured image by using an image synthesis and optimization algorithm. The processing unit analyzes and synthesizes the image to obtain three-dimensional light sheet imaging, including:

[0014] Step S1, multi-view image synthesis;

[0015] Step S2, deconvolution;

[0016] Step S3, shielding effect correction;

[0017] Step S4: 3D image reconstruction.

[0018] Preferably, the step S1 specifically includes:

[0019] Step S11, image registration: registering images at different viewing angles to align images at each viewing angle;

[0020] Step S12, image fusion: the registered multi-view images are fused at each pixel point;

[0021] Step S13, multi-channel fusion: the multi-view images of each channel are fused independently, and finally the fusion results of different channels are merged.

[0022] Preferably, the deconvolution algorithm used in step S2 is blind deconvolution, non-blind deconvolution or regularized deconvolution;

[0023] The shielding effect correction method used in step S3 is correction based on background modeling or correction based on multi-view fusion;

[0024] The three-dimensional image reconstruction algorithm used in step S4 is stacking reconstruction, reconstruction based on interpolation or volume rendering.

[0025] Preferably, a barrel mirror, a beam splitter and an objective lens are arranged between the camera and the target object. The camera is arranged perpendicular to the direction of the light sheet.

[0026] Preferably, the LED light source is a white LED or a RBG multicolor LED. The spatial light modulator is a DMD spatial light modulator, an LCOS spatial light modulator or a liquid crystal spatial light modulator. The projection imaging unit is a single lens or a multi-lens lens group.

[0027] The beneficial effects of the present invention are:

[0028] The light sheet microscopy system based on a spatial light modulator provided by the present invention generates a light sheet with a certain focal depth to illuminate the target through an LED light source, a spatial light modulator, and a projection imaging unit, and uses a camera to shoot the illuminated target layer in the vertical direction of the illumination, and performs data processing on the photographed image to realize light sheet imaging. The present invention can realize precise control of the light sheet position, can realize high-speed layer-by-layer scanning, can effectively avoid the influence of laser speckle and temperature drift, and has a simple optical path structure and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of a light sheet microscopy system based on a spatial light modulator according to the present invention.

[0030] In the figure: 1-LED light source; 2-beam shaping unit; 3-projection prism; 4-spatial light modulator; 5-projection imaging unit; 6-sample; 7-objective lens; 8-beam splitter; 9-barrel mirror; 10-camera. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention. The components of the embodiments of the present invention generally described and shown in the drawings herein may be arranged and designed in a variety of different configurations.

[0032] In the present invention, unless otherwise clearly stipulated and limited, terms such as "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal connection of two elements.

[0033] like Figure 1 As shown, a light sheet microscopy system based on a spatial light modulator of this embodiment includes a processing unit, an LED light source 1, a beam shaping unit 2, a projection prism 3, a spatial light modulator 4, a projection imaging unit 5, and a camera 10; wherein the LED light source 1 is a white LED or an RBG complex color LED, and the spatial light modulator 4 is a DMD spatial light modulator, an LCOS spatial light modulator, or a liquid crystal spatial light modulator.

[0034] The beam shaping unit 2 is used to shape the light emitted by the LED light source 1 into a uniform and collimated illumination beam, and irradiate the beam onto the spatial light modulator 4 through the projection prism 3 .

[0035] The projection imaging unit 5 is a single lens or a multi-lens lens group, and is used to shape the light field modulated by the spatial light modulator 4 into a light sheet that meets the requirements, and irradiate it onto the sample 6.

[0036] The camera 10 is arranged perpendicular to the direction of the light sheet. A barrel mirror 9, a beam splitter 8 and an objective lens 7 are arranged between the camera 10 and the target object. The camera 10 is used to collect the image of the sample 6 under the illumination of the light sheet.

[0037] The processing unit is used to encode the spatial light modulator 4, and to analyze and synthesize the captured image through an image synthesis and optimization algorithm to obtain three-dimensional light sheet imaging.

[0038] The working process of the light sheet microscopy system of this embodiment is as follows:

[0039] The light emitted by the LED light source 1 is shaped into a uniform and collimated illumination beam, and irradiated onto the spatial light modulator 4;

[0040] The spatial light modulator 4 is effectively encoded, and the light field modulated by the modulator is shaped into a light sheet that meets the requirements by using the projection imaging unit 5, and then irradiated to the sample 6;

[0041] Using a camera 10 arranged in a vertical direction to collect an image illuminated by the light sheet;

[0042] Repeat all the above steps, during which the spatial light modulator 4 is re-encoded and images are collected respectively, until the image collection of the entire sample 6 is completed;

[0043] The captured images are analyzed and synthesized using image synthesis and optimization algorithms to obtain the final three-dimensional light sheet imaging.

[0044] In this embodiment, in order to obtain high-quality three-dimensional reconstructed images, image synthesis and optimization algorithms play a vital role. The processing unit uses the image synthesis and optimization algorithms to analyze and synthesize the image to obtain three-dimensional light sheet imaging, including:

[0045] Step S1, multi-view image synthesis;

[0046] The image of the sample is collected from multiple angles. Each image at each angle is called a viewing angle. Multi-view imaging can obtain details of the sample in different directions, which helps to improve spatial resolution and reduce shielding effects. Synthesizing these multi-view images can significantly improve the quality of 3D reconstructed images. Specifically, it includes:

[0047] Step S11, image registration: images at different viewing angles may be displaced or rotated due to different imaging angles, and the images at different viewing angles are registered to align the images at each viewing angle; the image registration method is a registration based on feature points or a global registration algorithm based on mutual information or cross-correlation;

[0048] Step S12, image fusion: The registered multi-view images are fused at each pixel point, and the fusion process adopts the weighted average method. In order to avoid errors in edge areas, weights are allocated according to the distance from the optical axis. The area close to the optical axis has a larger weight, and the area far from the optical axis has a smaller weight.

[0049] Step S13, multi-channel fusion: the multi-view images of each channel are fused independently, and finally the fusion results of different channels are merged.

[0050] Step S2, deconvolution: Due to the optical characteristics of the system and the limitation of the light sheet thickness, the collected image is usually blurred. This blur mainly comes from the optical point spread function (PSF). Deconvolution can recover more details from the blurred image by inverting the PSF, thereby improving the resolution and contrast of the image.

[0051] The deconvolution algorithm is blind deconvolution, non-blind deconvolution or regularized deconvolution.

[0052] Blind Deconvolution: In this method, the PSF is not known in advance but is inferred by the algorithm. Blind deconvolution methods are adaptive filters, maximum likelihood estimation, etc.

[0053] Non-blind deconvolution: If the PSF is known (obtained through experimental calibration or theoretical calculation), non-blind deconvolution methods such as Richardson-Lucy Deconvolution or Wiener Filter can be directly applied.

[0054] Regularized deconvolution: Balance image detail restoration and noise suppression. Regularized deconvolution is Tikhonov regularization and total variation regularization.

[0055] Step S3, shading effect correction: Since this embodiment is a unidirectional lighting mode, the area close to the light source will produce a shadow or shading effect due to the scattering and absorption of light by the sample. This phenomenon will cause uneven brightness in local areas of the image, affecting the overall imaging quality. The shading effect correction algorithm reduces this unevenness by balancing the image brightness.

[0056] The occlusion effect correction method is correction based on background modeling or correction based on multi-view fusion.

[0057] Correction based on background modeling: By modeling the background brightness of the sample and analyzing the degree of light scattering and absorption, the distribution of the shadowing effect is estimated. Then, the brightness of the image is corrected based on this model to eliminate the shadowing effect.

[0058] Correction based on multi-view fusion: Through multi-view imaging, images at different viewpoints are affected differently by the occlusion effect. By fusing information from multiple viewpoints, the occlusion effect can be effectively mitigated.

[0059] Step S4, three-dimensional image reconstruction: the slice images are combined into a three-dimensional volume data, and then the three-dimensional structure of the sample is observed by three-dimensional reconstruction technology.

[0060] The 3D image reconstruction algorithms include stack reconstruction, interpolation-based reconstruction or volume rendering.

[0061] Stacking reconstruction: All slice images are stacked along the Z axis to generate a three-dimensional image. This method requires high image acquisition accuracy and must ensure that the spacing between each slice image is consistent.

[0062] Interpolation-based reconstruction: For slices with large acquisition intervals, interpolation methods can be used to generate more virtual slices to fill the gaps between images, thereby improving the resolution of 3D reconstruction. Common interpolation methods include bilinear interpolation and cubic spline interpolation.

[0063] Volume rendering: Volume rendering can generate visualization images directly from 3D volume data without explicitly extracting surfaces. Common volume rendering algorithms include ray casting and maximum intensity projection. These algorithms generate 3D images by calculating the ray path and intensity of each voxel in the volume data.

[0064] The noise in the imaging of the light sheet microscopy system of this embodiment mainly comes from the electronic noise and photon noise of the detector, as well as the autofluorescence of the sample itself. Noise suppression and image enhancement are the key to optimizing imaging quality.

[0065] The following noise suppression and image enhancement algorithms can be used:

[0066] Median filtering: suitable for removing high-frequency noise in images.

[0067] Gaussian filtering: It can effectively suppress high-frequency noise while retaining most of the details of the image, and achieves a smoothing effect by convolving the image with a Gaussian kernel.

[0068] Non-local means: Remove noise by analyzing similar pixel blocks in the image, which can retain more detailed information.

[0069] Hessian matrix enhancement: In order to enhance the structural features in the image (such as lines or surfaces), an enhancement method based on the Hessian matrix can be used. This method enhances the structural features in the image while suppressing noise by analyzing the second-order derivative information of the image.

[0070] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A light sheet microscopy system based on a spatial light modulator, characterized in that: include: LED light source (1); A beam shaping unit (2) is used to shape the light emitted by the LED light source (1) into a uniform and collimated illumination beam, and irradiate the beam onto the spatial light modulator (4) through a projection prism (3); A projection imaging unit (5) is used to shape the light field modulated by the spatial light modulator (4) into a light sheet that meets the requirements, and irradiate the light sheet onto a target object; A camera (10) collects an image of a target object under the illumination of the light sheet; The processing unit is used to encode the spatial light modulator (4) and to analyze and synthesize the captured image to obtain three-dimensional light sheet imaging.

2. The light sheet microscopy system according to claim 1, characterized in that: The processing unit analyzes and synthesizes the captured images through image synthesis and optimization algorithms.

3. The light sheet microscopy system according to claim 1 or 2, characterized in that: The processing unit analyzes and synthesizes the image to obtain three-dimensional light sheet imaging, including: Step S1, multi-view image synthesis; Step S2, deconvolution; Step S3, shielding effect correction; Step S4: 3D image reconstruction.

4. The light sheet microscopy system according to claim 3, characterized in that: The step S1 specifically includes: Step S11, image registration: registering images at different viewing angles to align images at each viewing angle; Step S12, image fusion: the registered multi-view images are fused at each pixel point; Step S13, multi-channel fusion: the multi-view images of each channel are fused independently, and finally the fusion results of different channels are merged.

5. The light sheet microscopy system according to claim 3, characterized in that: The deconvolution algorithm used in step S2 is blind deconvolution, non-blind deconvolution or regularized deconvolution; The shielding effect correction method used in step S3 is correction based on background modeling or correction based on multi-view fusion; The three-dimensional image reconstruction algorithm used in step S4 is stacking reconstruction, reconstruction based on interpolation or volume rendering.

6. The light sheet microscopy system according to claim 1, characterized in that: A barrel mirror (9), a beam splitter (8) and an objective lens (7) are provided between the camera (10) and the target object.

7. The light sheet microscopy system according to claim 1, characterized in that: The camera (10) is arranged perpendicular to the direction of the light sheet.

8. The light sheet microscopy system according to claim 1, characterized in that: The LED light source (1) is a white LED or an RBG multi-color LED.

9. The light sheet microscopy system according to claim 1, characterized in that: The spatial light modulator (4) is a DMD spatial light modulator, an LCOS spatial light modulator or a liquid crystal spatial light modulator.

10. The light sheet microscopy system according to claim 1, characterized in that: The projection imaging unit (5) is a single lens or a multi-lens lens group.

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