Super-resolution confocal microscopic imaging system

Abstract

The invention discloses a super-resolution confocal microscopic imaging system, which comprises a light modulation module, a spectrum splitting module, a scanning imaging module, an image detection device and a computer, and is characterized in that a light beam emitted by the light modulation module passes through the scanning imaging module and then enters a sample to excite a fluorescence signal; a fluorescence signal is received by the image detection device after passing through the spectrum splitting module, the image detection device converts a current signal into a digital signal and transmits the digital signal to the computer to complete imaging, the device is characterized in that the image detection device is an array detector composed of a plurality of sub-detectors, and after receiving the signal transmitted by each sub-detector, the computer sends the signal to the image detection device; according to the super-resolution reconstruction method, a detection point spread function is constructed for a dot matrix sub-graph of each sub-detector of the array detector, super-resolution information in the image is extracted, and super-resolution imaging is achieved. The super-resolution reconstruction method has the advantages that the super-resolution information in the image can be efficiently extracted, super-resolution imaging is achieved, and the imaging resolution is about 118 nm and is smaller than the diffraction limit.

Description

Technical Field

[0001] This invention relates to a confocal microscopy imaging system, and more particularly to a super-resolution confocal microscopy imaging system. Background Technology

[0002] Laser scanning confocal microscopy achieves high signal-to-noise ratio fluorescence imaging by adding a "conjugate pinhole" to the fluorescence optical path, resulting in a point-to-point image. In recent years, confocal microscopy has played an increasingly important role in biological and medical imaging fields due to its optical sectioning capabilities and high contrast. However, the resolution limit of traditional confocal microscopy is approximately 180 nm, which cannot meet researchers' needs for observing structures beyond the diffraction limit. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a super-resolution confocal microscopy imaging system with an imaging resolution of less than 120 nm, which can solve the problem of insufficient imaging resolution of traditional confocal microscopes.

[0004] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a super-resolution confocal microscopy imaging system, including a light modulation module, a spectral splitting module, a scanning imaging module, an image detection device, and a computer. The light beam emitted by the light modulation module passes through the scanning imaging module and is incident on the sample to excite a fluorescence signal. The fluorescence signal is received by the image detection device after passing through the spectral splitting module. The image detection device converts the current signal into a digital signal and transmits it to the computer to complete the imaging. The image detection device is an array detector composed of multiple sub-detectors. After receiving the signal transmitted by each sub-detector, the computer constructs a detection point spread function for each sub-detector of the array detector based on the detected high-frequency information carrying sample details through a super-resolution reconstruction method, extracts the super-resolution information in the image, and realizes super-resolution imaging.

[0005] Compared with existing technologies, the advantages of this invention are that, unlike traditional confocal systems that use "point scanning and point imaging", it can achieve "point scanning and multi-point imaging" by using an array detector composed of multiple sub-detectors. It can detect high-frequency information carrying sample details. In the back-end super-resolution reconstruction algorithm, by constructing a detection point diffusion function for each sub-image, the super-resolution information in the image can be efficiently extracted to achieve super-resolution imaging with an imaging resolution of approximately 118 nm, which is less than the diffraction limit.

[0006] Preferably, the specific steps of the super-resolution reconstruction method are as follows:

[0007] S1. The computer divides the input image into N sub-images according to the number of sub-detectors, where N is the number of sub-detectors;

[0008] S2. Subgraph denoising is achieved by convolving each subgraph with its corresponding point spread function (PSF).

[0009] S3. After performing a Fourier transform on the denoised sub-image to the frequency domain, determine the offset of the sub-image;

[0010] S4. Transform the subgraph of S3 to the spatial domain using Fourier transform and reconstruct it within the spatial domain;

[0011] S5. Overlay all the reconstructed sub-images according to their corresponding offsets to obtain the composite image;

[0012] S6. Perform noise reduction processing on the synthesized image to finally output a super-resolution image.

[0013] Preferably, the spectral splitting module comprises a first lens, a first pinhole, a zoom lens group, and a second lens arranged sequentially. The first lens focuses the fluorescence signal at the center of the first pinhole onto the zoom lens group, and the beam emitted from the zoom lens group is focused onto the image detection device by the second lens. Based on the traditional confocal microscopy imaging principle, using a zoom lens group instead of a fixed lens allows for the adaptation of objectives with different magnifications.

[0014] Preferably, the zoom lens group consists of a first liquid lens and a second liquid lens, and the distance between the first liquid lens and the second liquid lens remains constant. This liquid lens combination enables rapid zooming, adapts to objectives with different magnifications, and eliminates the need for mechanical adjustment of the zoom structure, thus solving the problems of slow response and wear associated with traditional mechanical zoom structures.

[0015] Preferably, the focal length of the first liquid lens is defined as f1, the focal length of the second liquid lens is defined as f2, and the distance between the first liquid lens and the second liquid lens is defined as L, then the following relationship is satisfied: f2 = L - f1.

[0016] Preferably, the scanning imaging module comprises a first dichroic mirror, a two-dimensional scanning device, a second reflecting mirror, a scanning lens, a tube mirror, an objective lens, and a sample stage for placing the sample, arranged sequentially. The first dichroic mirror transmits the light beam emitted by the light modulation module and reflects the fluorescence signal. The light beam emitted by the light modulation module is transmitted through the first dichroic mirror and scanned in two dimensions by the two-dimensional scanning device. The light beam is perpendicularly incident on the scanning lens through the second reflecting mirror, collimated into a parallel beam by the tube mirror, and converged onto the sample stage by the objective lens, exciting the sample on the sample stage to generate a fluorescence signal. The fluorescence signal sequentially passes through the objective lens, the tube mirror, the scanning lens, the second reflecting mirror, and the two-dimensional scanning device, collimated into parallel light, and reflected by the first dichroic mirror into the first lens.

[0017] Preferably, the scanning imaging module comprises a two-dimensional scanning device, a second dichroic mirror, a scanning lens, a tube mirror, an objective lens, and a sample stage for placing the sample, arranged sequentially. The second dichroic mirror reflects the light beam emitted by the light modulation module while transmitting fluorescence signals. The light beam emitted by the light modulation module is incident on the two-dimensional scanning device to achieve two-dimensional scanning of the beam. It is then reflected by the second dichroic mirror and incident perpendicularly on the scanning lens. After being collimated into a parallel beam by the tube mirror, it is converged by the objective lens onto the sample stage, exciting the sample on the sample stage to generate a fluorescence signal. The fluorescence signal passes sequentially through the objective lens, the tube mirror, and the scanning lens, and then is transmitted through the second dichroic mirror into the first lens. Since the fluorescence of biological samples is weak and the reflectivity of the scanning system is low, by designing a super-resolution confocal non-retrograde optical path, the sample fluorescence no longer passes through the two-dimensional scanning device and directly enters the back-end imaging system, which can improve the fluorescence detection efficiency of the system and reduce the phototoxicity of the laser and the sample. Attached Figure Description

[0018] Figure 1 This is a diagram of the super-resolution confocal scanning microscopy system provided in Embodiment 1 of the present invention;

[0019] Figure 2 Flowchart of the super-resolution confocal reconstruction algorithm;

[0020] Figure 3 Comparison of confocal and super-resolution confocal imaging results;

[0021] Figure 4 for Figure 3 Enlarged view of the confocal imaging result within the dashed box;

[0022] Figure 5 for Figure 3 Enlarged view of the super-resolution confocal imaging result within the dashed box;

[0023] Figure 6 for Figure 4 and Figure 5 Intensity curve distribution diagram of the dashed line;

[0024] Figure 7 This is a diagram of the super-resolution confocal non-retractable microscopic imaging system provided in Embodiment 2 of the present invention. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1:

[0028] Figure 1 The super-resolution confocal scanning microscopy system provided in this embodiment is shown in the diagram. The light modulation module consists of the following components: light source 1, multimode fiber 2, collimating lens 3, first reflecting mirror 4, beam shaping device 5, beam splitter prism 6, camera 7, third lens 8, second pinhole 9, and fourth lens 10; the scanning imaging module consists of the following components: first dichroic mirror 11-1- for transmitting the illumination beam emitted by light source 1 and reflecting fluorescence signal, two-dimensional scanning device 12, second reflecting mirror 13, scanning lens 14, tube lens 15, objective lens 16, and sample stage 17; the spectral splitting module consists of the following components: first lens 18, first pinhole 19, first liquid lens 20, second liquid lens 21, second lens 22, array detector 23, and computer 24.

[0029] Using the device of this embodiment, the imaging process is as follows:

[0030] The illumination beam emitted by light source 1 is collimated into parallel light by multimode fiber 2 and collimating lens 3. The parallel light beam passes through first reflecting mirror 4 to adjust its two-dimensional exit angle, making the beam parallel to the subsequent optical axis. After passing through beam shaping device 5, the two-dimensional distribution of the parallel light beam changes from Gaussian to flat-top, resulting in a more uniform light spot. The flat-top parallel light beam is split into two parts by beam splitter prism 6. One part of the beam is reflected and imaged onto camera 7 to monitor the spatial two-dimensional intensity distribution of the illumination beam in real time. The other part of the beam is transmitted through beam splitter prism 6 and converged at the center of second pinhole 9 by third lens 8. The pinhole 9 can filter out stray light introduced by the system. The light beam is collimated by the fourth lens 10 and incident on the first dichroic mirror 11-1. After transmission, it passes through the two-dimensional scanning device 12. The main function of the two-dimensional scanning device 12 is to realize the two-dimensional scanning of the light beam. The scanning light beam emitted from the two-dimensional scanning device 12 is reflected by the reflecting mirror 13 and incident perpendicularly on the scanning lens 14. It is collimated into a parallel light beam by the tube mirror 15 and converged on the sample stage 17 by the objective lens 16 to excite the fluorescence signal of the sample. The fluorescence signal passes through the objective lens 16, tube mirror 15, scanning lens 14, reflecting mirror 13, two-dimensional scanning device 12 and the first dichroic mirror 11-1 in sequence and is reflected into the first lens 18. The fluorescence signal is converged at the center of the first pinhole 19 by the first lens 18. The first pinhole 19 can effectively filter out stray light from the non-focal plane in the fluorescence signal. The fluorescence signal achieves rapid zooming by passing through the first liquid lens 20 and the second liquid lens 21. The distance L between the two liquid lenses is fixed, and the magnification is determined by the focal length ratio (or optical power ratio) of the two liquid lenses, as shown in Table 1.

[0031] Table 1

[0032]

[0033] Table 1 lists the focal lengths of the first and second liquid lenses and the magnification calculations of the zoom system when using 4×, 10×, 20×, 40×, 60×, and 100× objectives. This zoom system can be adapted to different objectives. The fluorescence signal passes through the zoom system and is converged to the array detector 23 by the second lens 22. The current signal of each sub-detector is converted into a digital signal and transmitted in parallel to the computer 24 to complete image acquisition. Super-resolution imaging is then achieved through backend super-resolution reconstruction. The super-resolution reconstruction method is as follows: Figure 2 As shown, after image input, the image is split into N sub-images according to the number of sub-detectors. Each sub-image is convolved with its corresponding point spread function (PSF) to achieve sub-image denoising. Then, it undergoes a Fourier transform to the frequency domain to determine the sub-image offset. Finally, it undergoes another Fourier transform to the spatial domain for spatial reconstruction. The sub-images are then superimposed according to their corresponding offsets. After denoising processing, the final output is a super-resolution image. The resolution comparison results of confocal and super-resolution confocal imaging are shown below. Figures 3-6 As shown, from Figure 6As can be seen from the intensity curve of the super-resolution confocal imaging, the imaging resolution of the super-resolution confocal microscopy system of this invention is about 118 nm, which is less than the diffraction limit.

[0034] Example 2:

[0035] Figure 7 This is a diagram of the super-resolution confocal non-retractable microscopic imaging system based on a liquid lens provided in this embodiment. Figure 7 As can be seen from the above, the light modulation module, spectral splitting module, image detection device 23 and computer 24 in Embodiment 2 are the same as those in Embodiment 1. The difference is that the scanning imaging module is composed of the following components: two-dimensional scanning device 12, second dichroic mirror 11-2 (used to reflect the illumination beam emitted by the light source 1 and transmit the fluorescence signal), scanning lens 14, tube lens 15, objective lens 16, and sample stage 17.

[0036] The imaging process using the device in this example is as follows:

[0037] The illumination beam emitted by light source 1 is collimated into parallel light by multimode fiber 2 and collimating lens 3. The parallel light beam passes through first reflecting mirror 4 to adjust its two-dimensional exit angle, making the beam parallel to the subsequent optical axis. After passing through beam shaping device 5, the two-dimensional distribution of the parallel light beam changes from Gaussian to flat-top, resulting in a more uniform light spot. The flat-top parallel light beam is split into two parts by beam splitter prism 6. One part of the beam is reflected and imaged onto camera 7 to monitor the spatial two-dimensional intensity distribution of the illumination beam in real time. The other part of the beam is transmitted through beam splitter prism 6 and converged at the center of second pinhole 9 by third lens 8. The pinhole 9 can filter out stray light introduced by the system. The light beam is collimated by the fourth lens 10 and incident on the two-dimensional scanning device 12 to achieve two-dimensional scanning of the beam. The scanning beam emitted from the two-dimensional scanning device 12 is reflected by the second dichroic mirror 11-2 and enters the scanning lens 14. It is collimated into a parallel beam by the tube mirror 15 and converged on the sample stage 17 by the objective lens 16. The beam excites the fluorescence signal of the sample. The fluorescence signal passes through the objective lens 16, the tube mirror 15, and the scanning lens 14, and is transmitted through the second dichroic mirror 11-2 into the spectral splitting module. In the spectral splitting module, the fluorescence signal is converged at the center of the first pinhole 19 by the first lens 18. 9 can effectively filter out stray light from the non-focal plane in the fluorescence signal. The fluorescence signal achieves rapid zooming through the first liquid lens 20 and the second liquid lens 21. The distance L between the two liquid lenses is fixed, and the magnification is determined by the focal length ratio (or optical power ratio) of the two liquid lenses to adapt to different objectives and realize the zooming of the detection system. The fluorescence signal passes through the zooming system and is converged to the array detector 23 by the second lens 22. The current signal of each sub-detector is converted into a digital signal and transmitted in parallel to the computer 24 to complete the imaging acquisition. After super-resolution reconstruction at the back end, super-resolution imaging is realized and a super-resolution image is output.

[0038] Compared to Example 1, this solution can improve the fluorescence detection efficiency of the entire super-resolution confocal system, reduce sample phototoxicity, and further expand the application range of the product.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A super-resolution confocal microscopy imaging system, comprising a light modulation module, a spectral splitting module, a scanning imaging module, an image detection device, and a computer, wherein a light beam emitted from the light modulation module passes through the scanning imaging module and is incident on a sample to excite a fluorescence signal; the fluorescence signal passes through the spectral splitting module and is received by the image detection device; the image detection device converts the current signal into a digital signal and transmits it to the computer to complete the imaging, characterized in that... The image detection device is an array detector composed of multiple sub-detectors. After receiving the signal transmitted by each sub-detector, the computer constructs a detection point spread function for each sub-detector's dot matrix image based on the detected high-frequency information carrying sample details using a super-resolution reconstruction method, extracts the super-resolution information in the image, and achieves super-resolution imaging.

2. The super-resolution confocal microscopy imaging system as described in claim 1, characterized in that, The specific steps of the super-resolution reconstruction method are as follows: S1. The computer divides the input image into N sub-images according to the number of sub-detectors, where N is the number of sub-detectors; S2. Subgraph denoising is achieved by convolving each subgraph with its corresponding point spread function (PSF). S3. After performing a Fourier transform on the denoised sub-image to the frequency domain, determine the offset of the sub-image; S4. Transform the subgraph of S3 to the spatial domain using Fourier transform and reconstruct it within the spatial domain; S5. Overlay all the reconstructed sub-images according to their corresponding offsets to obtain the composite image; S6. Perform noise reduction processing on the synthesized image to finally output a super-resolution image.

3. The super-resolution confocal microscopy imaging system as described in claim 1, characterized in that, The spectral splitting module consists of a first lens, a first pinhole, a zoom lens group, and a second lens arranged sequentially. The first lens focuses the fluorescence signal at the center of the first pinhole and directs it into the zoom lens group. The light beam emitted from the zoom lens group is then focused onto the image detection device by the second lens.

4. The super-resolution confocal microscopy imaging system as described in claim 3, characterized in that, The zoom lens group consists of a first liquid lens and a second liquid lens, and the distance between the first liquid lens and the second liquid lens remains constant.

5. The super-resolution confocal microscopy imaging system as described in claim 4, characterized in that, Let the focal length of the first liquid lens be f1, the focal length of the second liquid lens be f2, and the distance between the first liquid lens and the second liquid lens be L. Then the following relationship is satisfied: f2 = L - f1.

6. A super-resolution confocal microscopy imaging system as described in claim 3, 4, or 5, characterized in that, The scanning imaging module comprises a first dichroic mirror, a two-dimensional scanning device, a second reflecting mirror, a scanning lens, a tube mirror, an objective lens, and a sample stage for placing the sample, arranged sequentially. The first dichroic mirror transmits the light beam emitted by the light modulation module and reflects the fluorescence signal. The light beam emitted by the light modulation module is transmitted through the first dichroic mirror and scanned in two dimensions by the two-dimensional scanning device. The light beam is perpendicularly incident on the scanning lens by the second reflecting mirror, collimated into a parallel beam by the tube mirror, and converged onto the sample stage by the objective lens, exciting the sample on the sample stage to generate a fluorescence signal. The fluorescence signal sequentially passes through the objective lens, the tube mirror, the scanning lens, the second reflecting mirror, and the two-dimensional scanning device, collimated into parallel light, and reflected by the first dichroic mirror into the first lens.

7. A super-resolution confocal microscopy imaging system as described in claim 3, 4, or 5, characterized in that, The scanning imaging module consists of a two-dimensional scanning device, a second dichroic mirror, a scanning lens, a tube mirror, an objective lens, and a sample stage for placing the sample, arranged sequentially. The second dichroic mirror is used to reflect the light beam emitted by the light modulation module and transmit the fluorescence signal. The light beam emitted by the light modulation module is incident on the two-dimensional scanning device to achieve two-dimensional scanning of the beam. It is then reflected by the second dichroic mirror and incident perpendicularly on the scanning lens. After being collimated into a parallel beam by the tube mirror, it is focused by the objective lens onto the sample stage, exciting the sample on the sample stage to generate a fluorescence signal. The fluorescence signal passes sequentially through the objective lens, the tube mirror, and the scanning lens, and then is transmitted through the second dichroic mirror into the first lens.

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