A system and method for automatically finding the optimal oblique sheet light illumination position

Abstract

The application discloses a system and method for automatically searching for an optimal oblique layered light sheet illumination position, and belongs to the technical field of image data processing. The method comprises the following steps: exciting light is collimated and expanded, is adjusted by a first mirror to be incident to an internal light path of an electric displacement table, is reflected by a dichroic mirror to enter a TIRF objective, is focused by the TIRF objective, and is irradiated on a sample carried by a cover glass; the emitted light of the sample is collected by the TIRF objective, is transmitted by the dichroic mirror, is reflected by a second mirror, and enters a camera; a computer controls the electric displacement table to move, and acquires a fluorescence image collected by the camera; and a position point with the maximum fluorescence signal intensity value is determined as the optimal oblique layered light sheet illumination position. The application can automatically, quickly and accurately find the optimal oblique layered light sheet illumination position, avoids artificial subjective judgment errors, has the advantages of automation, self-adaptation and high accuracy, and significantly improves the experimental efficiency and quality of single-molecule localization super-resolution imaging.

Description

Technical Field

[0001] This invention relates to the field of image data processing technology, and in particular to a system and method for automatically finding the optimal illumination position of a tilted layered light sheet. Background Technology

[0002] In single-molecule localization super-resolution imaging systems, the illumination method is crucial for achieving sparse activation and high-quality imaging of single molecules. Traditional epi-irradiation (Epi) excites the entire sample through the objective lens, resulting in a strong defocused fluorescence background, which is detrimental to the separation and localization of single-molecule signals. While total internal reflection illumination (TIRF) can achieve excitation of extremely thin layers, its weak excitation light intensity and limited penetration depth restrict its application in observing intracellular structures. Therefore, neither of these two illumination methods can meet the illumination requirements of single-molecule localization super-resolution imaging.

[0003] Tilt-layer illumination (HILO), as an illumination method suitable for single-molecule localization super-resolution imaging, forms a thin sheet with a higher focused energy density by controlling the excitation light to be incident at an angle. This effectively suppresses defocused background fluorescence and achieves an effective imaging depth of about one micrometer. It balances excitation light intensity, signal-to-noise ratio and penetration depth, providing favorable conditions for sparse activation and precise localization of single-molecule signals.

[0004] However, in existing single-molecule localization super-resolution imaging systems, accurately locating the optimal tilted layered light sheet illumination position is difficult. It often relies on the experimenter's experience to manually adjust the motorized stage, changing the position of the excitation light entering the objective lens, and visually observing the total fluorescence image intensity acquired by the camera, repeatedly comparing and judging the positions of similar peak signals. This method is not only inefficient, highly subjective, and has poor repeatability, making it difficult to guarantee optimal single-molecule activation and imaging results every time, but it also requires a high level of operator experience, making it difficult for novices to achieve rapid and accurate localization.

[0005] Some commercially available single-molecule localization super-resolution imaging systems have preset illumination positions at the factory and visualize these positions through animation during adjustment, reducing the difficulty of user operation. However, the illumination positions of such systems are usually fixed parameters, relying on factory-set pre-defined conditions. They cannot adaptively adjust based on the thickness of different samples and slides in real experiments, making it difficult to consistently achieve optimal background suppression and signal activation in actual single-molecule localization imaging experiments, thus affecting the final super-resolution reconstruction quality. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a system and method for automatically finding the optimal illumination position of the tilted layered light sheet, which solves the problem that the illumination position of the tilted layered light sheet in the existing single-molecule positioning super-resolution imaging system depends on manual adjustment, which is inefficient and makes it difficult to find the optimal illumination position.

[0007] This invention is achieved through the following technical solution: A method for automatically finding the optimal illumination position of an inclined layered light sheet includes the following steps: S1: The excitation light emitted from the laser is collimated and expanded by the laser collimation and beam expanding module, and then adjusted by the first reflecting mirror to be incident on the internal optical path of the electric displacement stage. After the internal optical path of the electric displacement stage is adjusted, it is incident on the dichroic mirror, and then the dichroic mirror reflects the excitation light into the TIRF objective. The excitation light is focused by the TIRF objective to form a focused layered light sheet, which illuminates the sample supported by the coverslip. The emitted light generated by the sample after being excited is collected by the TIRF objective, and then transmitted by the dichroic mirror and reflected by the second reflecting mirror before entering the camera. S2: The computer controls the electric displacement stage to move step by step according to the preset step length, and simultaneously acquires the fluorescence image collected by the camera; S3: Calculate the average pixel intensity of the fluorescence image acquired by the camera as the fluorescence signal intensity value when the electric displacement stage moves to the current position, and determine the position with the largest fluorescence signal intensity value as the optimal tilted layered light sheet illumination position.

[0008] The optimized laser collimation and beam expanding module in step S1 includes a first plano-convex lens and a second plano-convex lens.

[0009] In the optimized version, the internal optical path of the electric displacement stage in step S1 includes a third reflecting mirror, a fourth reflecting mirror, and a third plano-convex lens. The excitation light reflected by the first reflecting mirror is vertically reflected to the third reflecting mirror, and after being reflected by the fourth reflecting mirror, it is horizontally emitted to the third plano-convex lens.

[0010] The optimized camera is an sCMOS camera suitable for single-molecule localization super-resolution imaging, with a stepping accuracy of 10 micrometers and a repeatability of 1 micrometer for the motorized displacement stage.

[0011] Furthermore, in step S2, the computer uses the following method to control the electric displacement stage to move step by step according to a preset step size: first, the electric displacement stage is controlled to perform a coarse scan with a large step size to locate the region with the maximum fluorescence signal intensity, and then a fine scan is performed with a small step size near the region with the maximum fluorescence signal intensity until the position of the maximum fluorescence signal intensity is accurately determined.

[0012] The optimized step size is 50 micrometers, and the small step size is 10 micrometers.

[0013] Furthermore, in step S3, the average pixel intensity of the fluorescence image acquired by the camera is calculated according to equation (1), and the calculated average pixel intensity is used as the fluorescence signal intensity value when the electric displacement stage moves to the current position: (1); in: This indicates the fluorescence signal intensity value as the electric displacement stage moves to its current position. Indicates the first The intensity value of each pixel. Indicates the first Background noise value per pixel This indicates the total number of pixels.

[0014] Furthermore, the computer automatically records the position of each electric displacement stage and its corresponding fluorescence signal intensity value, generates a position-intensity relationship curve, identifies the position of the peak point through the position-intensity relationship curve, and uses the peak point position as the optimal tilted layered light sheet illumination position.

[0015] A system for automatically finding the optimal illumination position of an inclined layered light sheet, for performing a method for automatically finding the optimal illumination position of an inclined layered light sheet as described in any of the above, comprising a laser, a laser collimating and expanding module, a first mirror, an electric displacement stage, a dichroic mirror, a TIRF objective lens, a coverslip, a second mirror, a camera, and a computer; The laser is used to emit excitation light; The laser collimation and beam expansion module is used to collimate and expand the excitation light; The first reflector is used to adjust the excitation light incident on the electric displacement stage; The electric displacement stage is equipped with an internal optical path for adjusting the position of the excitation light incident on the TIRF objective lens; The dichroic mirror is located at the intersection of the excitation light and the emission light, and is used to reflect the excitation light into the TIRF objective lens and transmit the emission light generated after the sample is excited to the second mirror. The TIRF objective lens is used to receive excitation light, then converge the excitation light to form a focused layered light sheet, which illuminates the sample supported by the coverslip, and collects the emitted light generated after the sample is excited, which is then directed to the dichroic mirror. The second reflector is used to reflect the emitted light transmitted by the dichroic mirror into the camera; The camera is used to acquire fluorescence images; The computer is used to control the electric displacement stage to move step by step according to a preset step size and to perform data processing.

[0016] The optimized laser collimation and beam expansion module includes a first plano-convex lens and a second plano-convex lens. The internal optical path of the electric displacement stage includes a third reflecting mirror, a fourth reflecting mirror, and a third plano-convex lens. The excitation light reflected by the first reflecting mirror is vertically reflected to the third reflecting mirror, and after being reflected by the fourth reflecting mirror, it is horizontally emitted to the third plano-convex lens.

[0017] Beneficial effects of the invention: By connecting a computer to an electric displacement stage and a camera, and controlling their coordinated operation, the excitation illumination position can be automatically adjusted, thereby quickly and accurately finding the optimal tilted layered light sheet illumination position. This avoids errors caused by subjective human judgment and has the advantages of automation, self-adaptation, and high accuracy. It significantly improves the reconstructed image quality of single-molecule localization super-resolution imaging, and the system does not require the introduction of additional devices, resulting in relatively low cost. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the process of this invention.

[0019] Figure 2a This is a comparative diagram of tilted layered light sheet illumination (HILO) and traditional incident illumination (Epi) and total internal reflection illumination (TIRF).

[0020] Figure 2b This is a schematic diagram of the sample under HILO (High-Intensity Loop) illumination.

[0021] Figure 3a This is a curve showing the relationship between position and intensity obtained during the coarse scanning process of this invention.

[0022] Figure 3b This is a curve showing the relationship between position and intensity obtained during the fine scanning process of this invention.

[0023] Figure 4 This is a schematic diagram of the system of the present invention.

[0024] In the figure: 1. Laser; 2. First plano-convex lens; 3. Second plano-convex lens; 4. First reflecting mirror; 5. Third reflecting mirror; 6. Fourth reflecting mirror; 7. Third plano-convex lens; 8. Electric displacement stage; 9. Dichroic mirror; 10. TIRF objective lens; 11. Cover glass; 12. Sample; 13. Second reflecting mirror; 14. Camera; 15. Computer. Detailed Implementation

[0025] A method for automatically finding the optimal illumination position of an inclined layered light sheet is illustrated in the flowchart below. Figure 1 As shown, it includes the following steps: S1: The excitation light emitted from the laser is collimated and expanded by the laser collimation and beam expanding module, and then adjusted by the first reflecting mirror to be incident on the internal optical path of the electric displacement stage. After the internal optical path of the electric displacement stage is adjusted, it is incident on the dichroic mirror, and then the dichroic mirror reflects the excitation light into the TIRF objective. The excitation light is focused by the TIRF objective to form a focused layered light sheet, which illuminates the sample supported by the coverslip. The emitted light generated by the sample after being excited is collected by the TIRF objective, and then transmitted by the dichroic mirror and reflected by the second reflecting mirror before entering the camera. The laser collimation and beam expansion module may include a first plano-convex lens and a second plano-convex lens, which together form a whole to collimate and expand the excitation light.

[0026] The internal optical path of the motorized stage includes a third reflecting mirror, a fourth reflecting mirror, and a third plano-convex lens. The excitation light reflected by the first reflecting mirror is vertically reflected to the third reflecting mirror, and then reflected horizontally by the fourth reflecting mirror before exiting to the third plano-convex lens. The motorized stage carries the third reflecting mirror, the fourth reflecting mirror, and the third plano-convex lens. By moving the motorized stage, the position of the excitation light incident on the TIRF objective lens can be adjusted.

[0027] The electric displacement stage can be any programmable high-precision, high-repeatability displacement stage. The stepping accuracy of the electric displacement stage can preferably be 10 micrometers, and the repeatability can preferably be 1 micrometer.

[0028] The preferred camera is an sCMOS camera suitable for single-molecule localization super-resolution imaging. sCMOS cameras are the preferred choice for applications in high-throughput fluorescence imaging, microscopic imaging, cold atom or quantum research, space debris monitoring, and solar astronomy. They feature high resolution, ultra-low noise, large format, high performance, high sensitivity, low noise, and high frame rate, making them even more suitable for single-molecule localization super-resolution imaging.

[0029] S2: The computer controls the electric displacement stage to move step by step according to the preset step length, and simultaneously acquires the fluorescence image collected by the camera; Specifically, the computer can control the electric displacement stage to move step by step according to a preset step size using the following method: First, control the electric displacement stage to perform a coarse scan with a large step size to locate the region with the maximum fluorescence signal intensity. Then, perform a fine scan with a small step size near the region with the maximum fluorescence signal intensity until the position of the maximum fluorescence signal intensity is accurately determined.

[0030] Here, the larger step size can be preferably 50 micrometers, and the smaller step size can be preferably 10 micrometers.

[0031] The computer-controlled electric displacement stage moves stepwise along the optical path. At each set position, the sample is irradiated with excitation light to stimulate fluorescence emission. The fluorescence signal is then acquired by a camera to generate a corresponding fluorescence image.

[0032] By first controlling the electric displacement stage to perform a coarse scan with a large step size, the region with the maximum fluorescence signal intensity can be quickly located. Then, a fine scan with a small step size can be performed near the region with the maximum fluorescence signal intensity, which can further improve the speed of finding the location of the maximum fluorescence signal intensity.

[0033] S3: Calculate the average pixel intensity of the selected area of ​​the fluorescence image acquired by the camera as the fluorescence signal intensity value when the electric displacement stage moves to the current position, and determine the position with the largest fluorescence signal intensity value as the optimal tilted layered light sheet illumination position.

[0034] Specifically, the computer can calculate the average pixel intensity of the fluorescence image acquired by the camera according to equation (1), and use the calculated average pixel intensity as the fluorescence signal intensity value when the electric displacement stage moves to the current position: (1); in: This indicates the fluorescence signal intensity value as the electric displacement stage moves to its current position. Indicates the first The intensity value of each pixel. Indicates the first Background noise value per pixel This indicates the total number of pixels.

[0035] The average pixel intensity of the selected area of ​​the fluorescence image acquired by the camera is calculated according to formula (1). The calculated average pixel intensity is used as the fluorescence signal intensity value when the electric displacement stage moves to the current position. The background noise of the original fluorescence image acquired by the camera can be subtracted to obtain the net fluorescence signal image, making the found optimal tilted layered light sheet illumination position more accurate.

[0036] In the process of finding the optimal tilted layered light sheet illumination position, the computer can automatically record the position of each motorized displacement stage and its corresponding fluorescence signal intensity value, generate a position-intensity relationship curve, identify the position of the peak point through the position-intensity relationship curve, and take the position of the peak point as the optimal tilted layered light sheet illumination position.

[0037] This method achieves objective and accurate determination of lighting position through automated scanning and intensity quantification analysis, effectively avoiding subjective errors caused by manual adjustment.

[0038] A comparative diagram of specific tilted layered light sheet illumination (HILO) with traditional epi-irradiated illumination (Epi) and total internal reflection illumination (TIRF) is shown below. Figure 2a As shown, a schematic diagram of the sample under tilted layered light sheet illumination (HILO) is presented. Figure 2b As shown. From Figure 2a and Figure 2bIt can be seen that the tilted layered light sheet illumination method can more accurately excite the focal plane of the sample and its adjacent areas, significantly reducing the defocus noise signal outside the imaging depth, and is suitable for the signal-to-noise ratio requirements of single-molecule localization super-resolution imaging.

[0039] The position-intensity relationship curve obtained when a computer-controlled electric displacement stage performs coarse scanning with a large step size of 50 micrometers is shown below. Figure 3a As shown, by Figure 3a It can be seen that the average pixel intensity is relatively high around 5.2 to 5.3 mm. Therefore, the region with the maximum fluorescence signal intensity is determined to be within the range of 5.1 to 5.5 mm. Then, fine scanning is performed within the range of 5.1 to 5.5 mm using a small step size. The obtained position-intensity relationship curve is shown below. Figure 3b As shown. By Figure 3b It can be seen that after multiple round-trip scans by the electric displacement stage, the peak positions of the obtained positions and intensity curves have a high degree of overlap, and the fluctuation range of the values ​​is very small. This indicates that the method for automatically finding the optimal illumination position of the tilted layered light sheet provided by this invention, with its efficient linkage between the electric displacement stage and the camera and adaptive step size, can achieve non-destructive and rapid scanning of the sample, avoiding quenching, bleaching, and other effects on the sample itself. This significantly improves the stability and repeatability of the system, and the system does not require the introduction of additional devices, resulting in low cost.

[0040] A system for automatically finding the optimal illumination position of an inclined layered light sheet is provided for performing a method for automatically finding the optimal illumination position of an inclined layered light sheet as described in any of the above embodiments. A schematic diagram of the system is shown below. Figure 4 As shown, it includes a laser 1, a laser collimation and beam expansion module, a first reflecting mirror 4, an electric displacement stage 8, a dichroic mirror 9, a TIRF objective lens 10, a coverslip 11, a second reflecting mirror 13, a camera 14, and a computer 15. The laser is used to emit excitation light; The laser collimation and beam expansion module is used to collimate and expand the excitation light; The first reflector is used to adjust the excitation light incident on the electric displacement stage; The electric displacement stage is equipped with an internal optical path for adjusting the position of the excitation light incident on the TIRF objective lens; The dichroic mirror is located at the intersection of the excitation light and the emission light, and is used to reflect the excitation light into the TIRF objective lens and transmit the emission light generated after the sample 12 is excited to the second mirror. The TIRF objective lens is used to receive excitation light, and then converges the excitation light to form a focused layered light sheet, which illuminates the sample supported by the coverslip, and directs the emitted light generated by the sample after excitation to the dichroic mirror. The second reflector is used to reflect the emitted light transmitted by the dichroic mirror into the camera; The camera is used to acquire fluorescence images; The computer is used to control the electric displacement stage to move step by step according to a preset step size and to perform data processing.

[0041] The optimized laser collimation and beam expansion module includes a first plano-convex lens 2 and a second plano-convex lens 3. The internal optical path of the electric displacement stage includes a third reflecting mirror 5, a fourth reflecting mirror 6 and a third plano-convex lens 7. The excitation light reflected by the first reflecting mirror is vertically reflected to the third reflecting mirror, and after being reflected by the fourth reflecting mirror, it is horizontally emitted to the third plano-convex lens.

[0042] In summary, the system and method for automatically finding the optimal tilted layered light sheet illumination position provided by this invention can automatically adjust the excitation light illumination position without introducing additional devices, thereby quickly and accurately finding the optimal tilted layered light sheet illumination position, avoiding errors caused by subjective human judgment, and has the advantages of automation, adaptability, and high accuracy, significantly improving the reconstructed image quality of single-molecule localization super-resolution imaging.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for automatically finding the optimal illumination position of an inclined layered light sheet, characterized in that: The steps include the following: S1: The excitation light emitted from the laser is collimated and expanded by the laser collimation and beam expanding module, and then adjusted by the first reflecting mirror to be incident on the internal optical path of the electric displacement stage. After adjustment by the internal optical path of the electric displacement stage, it is incident on the dichroic mirror, and then the dichroic mirror reflects the excitation light into the TIRF objective. The excitation light is focused by the TIRF objective to form a focused layered light sheet, which illuminates the sample supported by the cover glass. The emitted light generated after the sample is excited is collected by the TIRF objective, and then transmitted by the dichroic mirror and reflected by the second reflecting mirror before entering the camera. The internal optical path of the electric displacement stage includes a third reflecting mirror, a fourth reflecting mirror and a third plano-convex lens. The excitation light reflected by the first reflecting mirror is vertically reflected to the third reflecting mirror, and after being reflected by the fourth reflecting mirror, it is horizontally emitted to the third plano-convex lens. S2: The computer controls the electric displacement stage to move step by step according to the preset step length, and simultaneously acquires the fluorescence image collected by the camera; S3: Calculate the average pixel intensity of the fluorescence image acquired by the camera as the fluorescence signal intensity value when the electric displacement stage moves to the current position, and determine the position with the largest fluorescence signal intensity value as the optimal tilted layered light sheet illumination position.

2. The method for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 1, characterized in that: The laser collimation and beam expansion module in step S1 includes a first plano-convex lens and a second plano-convex lens.

3. The method for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 1, characterized in that: The camera is an sCMOS camera suitable for single-molecule localization super-resolution imaging, with a stepping accuracy of 10 micrometers and a repeatability of 1 micrometer for the motorized displacement stage.

4. The method for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 1, characterized in that: In step S2, the computer controls the electric displacement stage to move step by step according to a preset step size using the following method: First, the electric displacement stage is controlled to perform a coarse scan with a large step size to locate the region with the maximum fluorescence signal intensity. Then, a fine scan is performed near the region with the maximum fluorescence signal intensity using a small step size until the position of the maximum fluorescence signal intensity is accurately determined.

5. The method for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 4, characterized in that: The large step size is 50 micrometers, and the small step size is 10 micrometers.

6. The method for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 1, characterized in that: In step S3, the average pixel intensity of the fluorescence image acquired by the camera is calculated according to equation (1), and the calculated average pixel intensity is used as the fluorescence signal intensity value when the electric displacement stage moves to the current position: （1）； in: This indicates the fluorescence signal intensity value as the electric displacement stage moves to its current position. Indicates the first The intensity value of each pixel. Indicates the first Background noise value per pixel This indicates the total number of pixels.

7. The method for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 1, characterized in that: The computer automatically records the position of each electric displacement stage and its corresponding fluorescence signal intensity value, generates a position-intensity relationship curve, identifies the position of the peak point through the position-intensity relationship curve, and uses the position of the peak point as the optimal illumination position of the tilted layered light sheet.

8. A system for automatically finding the optimal illumination position of an inclined layered light sheet, used to perform a method for automatically finding the optimal illumination position of an inclined layered light sheet as described in any one of claims 1 to 7, characterized in that, It includes a laser, a laser collimation and beam expander module, a first reflecting mirror, an electric displacement stage, a dichroic mirror, a TIRF objective lens, a coverslip, a second reflecting mirror, a camera, and a computer; The laser is used to emit excitation light; The laser collimation and beam expansion module is used to collimate and expand the excitation light. The laser collimation and beam expansion module includes a first plano-convex lens and a second plano-convex lens. The internal optical path of the electric displacement stage includes a third reflecting mirror, a fourth reflecting mirror and a third plano-convex lens. The excitation light reflected by the first reflecting mirror is vertically reflected to the third reflecting mirror, and after being reflected by the fourth reflecting mirror, it is horizontally emitted to the third plano-convex lens. The first reflector is used to adjust the excitation light incident on the electric displacement stage; The electric displacement stage is equipped with an internal optical path for adjusting the position of the excitation light incident on the TIRF objective lens; The dichroic mirror is located at the intersection of the excitation light and the emission light, and is used to reflect the excitation light into the TIRF objective lens and transmit the emission light generated after the sample is excited to the second mirror. The TIRF objective lens is used to receive excitation light, then converge the excitation light to form a focused layered light sheet, and collect the emitted light generated after the sample is excited, and direct the emitted light to the dichroic mirror. The second reflector is used to reflect the emitted light transmitted by the dichroic mirror into the camera; The camera is used to acquire fluorescence images; The computer is used to control the electric displacement stage to move step by step according to a preset step size and to perform data processing.

Images