A disk scanning confocal illumination intensity adjustment method and apparatus

Abstract

This invention discloses an illumination intensity modulation method for disk scanning confocal imaging, comprising the following steps: S1: Before formal imaging, a pre-imaging is performed using weak, uniform illumination; S2: The fluorescence intensity of different regions in the pre-imaging is evaluated and analyzed, and the evaluation results are obtained; S3: Based on the evaluation results, the parameter settings of the intensity modulation mechanism are adjusted to achieve different illumination in different regions of the sample; S4: The scanning points traverse the imaging area to obtain a complete image on the camera. This invention can dynamically adjust the illumination intensity of local regions in the scanning imaging as needed, and can be used to reduce photobleaching in local regions, increase the dynamic range of imaging, and enhance the signal-to-noise ratio of local regions.

Description

Technical Field

[0001] This invention belongs to the field of optical instruments, specifically relating to a method and apparatus for adjusting illumination intensity using disk scanning confocal focusing. Background Technology

[0002] A rotary confocal imaging system places a Nipkow turntable on the image plane of the object. The turntable is covered with pinholes arranged in an Archimedean spiral. The laser source covers the area of ​​all the pinholes. When the turntable rotates through a certain angle, the scanning points can cover the entire imaging area. Compared with the traditional point scanning method, this scanning method greatly improves the acquisition speed.

[0003] For actual biological samples, different regions have different fluorescent molecule densities. If the same illumination light is used, some areas will saturate on the camera due to excessively high light intensity, while other areas will have a low signal-to-noise ratio due to insufficient light intensity. Point-scanning confocal microscopy can adjust the light intensity based on the light intensity of each imaging point using a high-speed light intensity adjustment mechanism. Rotary confocal microscopy, on the other hand, uses a uniform laser beam to illuminate the entire sample area and cannot modulate the light intensity of local areas. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0005] This invention provides a method for adjusting illumination intensity using disk scanning confocal imaging, comprising the following steps:

[0006] S1: Before the actual imaging, a pre-imaging is performed using weak, uniform illumination;

[0007] S2: Evaluate and analyze the fluorescence intensity of different regions in the pre-image and obtain the evaluation and analysis results;

[0008] S3: Adjust the parameter settings of the light intensity modulation mechanism according to the evaluation and analysis results to achieve different light intensity illumination in different areas of the sample;

[0009] S4: Scan points traverse the imaging area to obtain a complete image on the camera.

[0010] Furthermore, in multi-frame imaging of living organisms, due to changes in sample fluorescence intensity caused by biological activities, the image of the previous frame is used as the basis for regulating the fluorescence intensity of the region in the next imaging.

[0011] Another aspect of the present invention provides a disk scanning confocal illumination intensity modulation device, which includes a laser machine, a beam expander, an intensity modulation structure, a lens turntable, a beam splitter, a pinhole turntable, a lens, an objective lens, and a sample arranged sequentially along the optical path. The reflected light from the beam splitter passes through the lens to the camera, and the intensity modulation structure and the camera are connected to a control system.

[0012] Furthermore, the light intensity modulation mechanism is a digital micromirror or a spatial light modulator.

[0013] Furthermore, the beam splitter is a dichroic mirror that transmits short wavelengths and reflects long wavelengths.

[0014] This invention can dynamically adjust the illumination intensity of local areas in scanning imaging as needed, which can be used to reduce photobleaching in local areas, increase the dynamic range of imaging, and enhance the signal-to-noise ratio of local areas.

[0015] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the structure of an embodiment of the present invention.

[0018] Figure 2 This is a flowchart illustrating the intensity modulation process according to an embodiment of the present invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not imply the presence or addition of one or more other elements or combinations thereof.

[0021] The technical solution of the present invention will now be described in conjunction with the accompanying drawings.

[0022] like Figure 1 As shown, the present invention provides a disk scanning confocal illumination intensity modulation device, which includes a laser machine 1, a beam expander 2, an intensity modulation structure 3, a lens turntable 4, a beam splitter 5, a pinhole turntable 6, a lens 7, an objective lens 8, and a sample 9 arranged sequentially along the optical path. The reflected light from the beam splitter 5 passes through the lens 7 to the camera 10. The intensity modulation structure 3 and the camera 10 are connected to the control system.

[0023] The illumination light emitted by the laser is expanded by a beam expander and then projected onto a light intensity modulation mechanism. The light intensity modulation mechanism modulates the intensity of the illumination light. After modulation, the illumination light shines on the lens turntable and then converges onto the pinhole turntable. The illumination light passing through the pinhole illuminates the sample after passing through the lens and objective lens. The fluorescent molecules on the sample are excited and emit fluorescence. This fluorescence passes through the objective lens, lens, and pinhole turntable, and is finally reflected onto the camera by a dichroic mirror. As the lens turntable and pinhole turntable rotate, the scanning point traverses the imaging area, and a complete image is obtained on the camera.

[0024] In the embodiments provided by the present invention, optionally, the light intensity modulation mechanism 3 is a digital micromirror or a spatial light modulator.

[0025] In the embodiments provided by the present invention, optionally, the beam splitter 5 is a dichroic mirror that transmits short wavelengths and reflects long wavelengths.

[0026] When the illumination intensity is unsaturated, the fluorescence intensity is linearly related to the illumination intensity. Therefore, by combining the light intensity modulation parameters and the imaging results of the camera, the true fluorescence intensity distribution of the sample can be reconstructed.

[0027] Evaluation method: Generally, before saturation illumination is reached, the fluorescence intensity emitted by a fluorescent substance after excitation is linearly proportional to the excitation light intensity. Therefore, pre-scanning can be used to evaluate the fluorescence intensity of the sample and predict the fluorescence intensity under enhanced illumination. This can be divided into several cases:

[0028] (1) If the area with strong fluorescence intensity will saturate on the camera after the illumination intensity is increased, the illumination intensity of the area can be reduced while ensuring a certain signal-to-noise ratio. After imaging, the image intensity of the area can be multiplied by a parameter. For the final image, the dynamic range of the whole image is greater than that of the image with uniform illumination, and the weaker laser can reduce photobleaching.

[0029] (2) In areas with weak fluorescence intensity, the illumination intensity of the area can be appropriately increased to improve the imaging signal-to-noise ratio.

[0030] Specific adjustment methods are as follows Figure 2 As shown, it includes the following steps:

[0031] S1: Before the actual imaging, a pre-imaging is performed using weak, uniform illumination;

[0032] S2: Evaluate and analyze the fluorescence intensity of different regions in the pre-image and obtain the evaluation and analysis results;

[0033] S3: Adjust the parameter settings of the light intensity modulation mechanism according to the evaluation and analysis results to achieve different light intensity illumination in different areas of the sample;

[0034] S4: The scanning points traverse the imaging area to obtain a complete image on the camera. In the embodiments provided by this invention, in multi-frame imaging of a living organism, due to changes in the fluorescence intensity of the sample caused by biological activities, the image of the previous frame is used as the basis for fluorescence intensity modulation of the area in the next imaging.

[0035] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be easily implemented by those skilled in the art. Therefore, the present invention is not limited to the specific embodiments described above; the specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art, under the guidance of the present invention, can make many other modifications without departing from the spirit and scope of the claims, all of which are within the protection scope of the present invention.

Claims

1. A method of adjusting the intensity of illumination light for disc scanning confocal microscopy, characterized by, The method comprises the following steps: S1: before formal imaging, a pre-imaging is performed by weak uniform light illumination; S2: the fluorescence intensity of different regions of the pre-imaging is evaluated and analyzed, and the evaluation and analysis result is obtained, the evaluation and analysis is based on that the fluorescence intensity emitted by the fluorescent substance after excitation is linearly proportional to the excitation light intensity before saturation illumination is reached, so the fluorescence intensity of the sample is evaluated by pre-scanning, and the fluorescence intensity under the condition of enhanced intensity is predicted; S3: the parameter setting of the light intensity modulation mechanism is adjusted according to the evaluation and analysis result, so as to realize different light intensity illumination in different regions of the sample, and the purpose of adjusting the parameter setting is to reduce the local area light bleaching, increase the imaging dynamic range, and enhance the signal-to-noise ratio of the local area; S4: the scanning points traverse the imaging area, and a complete image is obtained on the camera.

2. The method of claim 1, wherein the step of adjusting the intensity of the illumination light is performed by a disk scanning confocal microscope. In the multi-frame imaging of living body, due to the change of sample fluorescence intensity caused by life activities, the image of the last frame is used as the basis for fluorescence light intensity modulation of the region of the next imaging.

3. A disk scanning confocal illumination intensity adjustment device, characterized in that, The disc scanning confocal illumination light intensity adjustment device comprises: A laser machine, a beam expander, a light intensity modulation structure, a lens turntable, a beam splitter, a pinhole turntable, a lens, an objective lens, and a sample are sequentially arranged along the light path direction, the reflected light of the beam splitter passes through the lens to the camera, and the light intensity modulation mechanism and the camera are connected with the control system.

4. A device for adjusting the intensity of the illumination light in a disc scanning confocal microscope as claimed in claim 3, characterized in that The light intensity modulation mechanism is a digital micro-mirror or a spatial light modulator.

5. The apparatus for adjusting the intensity of the illumination light for disc scanning confocal microscopy according to claim 3, wherein The beam splitter is a dichroic mirror which transmits short-wavelength light and reflects long-wavelength light. The beam splitter is a dichroic mirror which transmits short-wavelength light and reflects long-wavelength light.

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