System and method for automatically searching optimal inclined layered light sheet illumination position

Abstract

The invention discloses a system and a method for automatically searching an optimal inclined 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 reflector to enter an internal light path of an electric displacement table, then enters a dichroscope, is reflected by the dichroscope to enter a TIRF objective lens, is focused by the TIRF objective lens, and is irradiated on a sample carried by a cover glass; emitted light emitted by the sample is collected by the TIRF objective lens, transmitted by the dichroscope and reflected by the second reflector to enter the camera; the computer controls the electric displacement table to move to obtain a fluorescence image collected by the camera; and determining the position point with the maximum fluorescence signal intensity value as the optimal inclined layered light sheet illumination position. The method can automatically, quickly and accurately find the optimal inclined layered light sheet illumination position, avoids manual subjective judgment errors, has the advantages of automation, self-adaption and high accuracy, and remarkably improves the experimental efficiency and quality of single-molecule positioning super-resolution imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of image data processing, and in particular to a system and method for automatically finding an optimal oblique lamellar light sheet illumination position. BACKGROUND

[0002] In a single molecule localization super-resolution imaging system, the illumination method is crucial for achieving sparse activation of single molecules and high-quality imaging. Traditional epi-illumination (Epi) excites the sample as a whole through the objective lens, resulting in strong out-of-focus fluorescence background, which is not conducive to the separation and localization of single molecule signals. Although total internal reflection illumination (TIRF) can achieve extremely thin layer excitation, its weak excitation light intensity and limited penetration depth limit its application in observing internal structures of cells. Therefore, both of the above-mentioned illumination methods are difficult to meet the illumination needs of single molecule localization super-resolution imaging.

[0003] As an illumination method suitable for single molecule localization super-resolution imaging, oblique lamellar light sheet illumination (HILO) forms a thin layer of light sheet with high focusing energy density by controlling the excitation light to enter at an oblique angle, which effectively suppresses out-of-focus background fluorescence and achieves an effective imaging depth of about one micrometer, thereby balancing the excitation light intensity, signal-to-noise ratio and penetration depth, and providing favorable conditions for sparse activation and precise localization of single molecule signals.

[0004] However, in existing single molecule localization super-resolution imaging systems, the optimal oblique lamellar light sheet illumination position is difficult to accurately locate, and it is often manually adjusted by experimenters depending on their experience. The motorized displacement stage is adjusted to change the position of the excitation light entering the objective lens, and the total fluorescence image intensity collected by the camera is observed by the naked eye, and similar peak signal positions are repeatedly compared and judged. This method is not only inefficient, subjective and poorly reproducible, but also difficult to ensure optimal single molecule activation and imaging results every time. At the same time, it requires high experience of the operator, and it is difficult for beginners to achieve rapid and accurate positioning.

[0005] Some commercial single molecule localization super-resolution imaging systems have a pre-set light sheet illumination position when they are shipped, and the light sheet position is visualized in the form of animation during adjustment, which reduces the difficulty of user operation. However, the illumination position of such systems is usually a fixed parameter, which depends on the prior conditions at the time of shipment and cannot be adjusted adaptively according to the thickness of different samples, the thickness of the glass slide, etc. in real experiments, resulting in difficulty in obtaining the best background suppression and signal activation effect in actual single molecule localization imaging experiments, which affects the quality of the final super-resolution reconstruction. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a system and method for automatically searching for an optimal oblique layered light sheet illumination position, which solves the problem of low efficiency and difficulty in searching for an optimal illumination position in the prior art single molecule localization super-resolution imaging system, which relies on manual adjustment of the oblique layered light sheet illumination position.

[0007] The present application is implemented by the following technical solutions: The method for automatically searching for an optimal oblique layered light sheet illumination position comprises the following steps: S1: Excitation light emitted by a laser passes through a laser collimation and expansion module for collimation and expansion, is adjusted by a first mirror to enter an internal optical path of an electric displacement table, is adjusted by the internal optical path of the electric displacement table, is reflected by a dichroic mirror into a TIRF objective, and is focused by the TIRF objective to form a focused layered light sheet, which irradiates a sample carried by a cover glass. Emission light generated by the sample after excitation is collected by the TIRF objective, is transmitted by the dichroic mirror, is reflected by a second mirror, and enters a camera; S2: A computer controls the electric displacement table to move step by step at a preset step length, and synchronously acquires fluorescence images collected by the camera; S3: The average pixel intensity of the fluorescence images collected by the camera is calculated as a fluorescence signal intensity value when the electric displacement table moves to a current position, and a position point with the maximum fluorescence signal intensity value is determined as the optimal oblique layered light sheet illumination position.

[0008] Optimally, the laser collimation and expansion module in step S1 comprises a first plano-convex lens and a second plano-convex lens.

[0009] Optimally, the internal optical path of the electric displacement table in step S1 comprises a third mirror, a fourth mirror and a third plano-convex lens. The excitation light reflected by the first mirror is vertically reflected to the third mirror, is reflected by the fourth mirror, and is horizontally emitted to the third plano-convex lens.

[0010] Optimally, the camera is an sCMOS camera suitable for single molecule localization super-resolution imaging, the step-by-step accuracy of the electric displacement table is 10 microns, and the repeat positioning accuracy is 1 micron.

[0011] Further, the computer controls the electric displacement table to move step by step at a preset step length in step S2 by using the following method: first, the electric displacement table is controlled to perform coarse scanning at a large step length to position to a region with the maximum fluorescence signal intensity value, then fine scanning is performed at a small step length near the region with the maximum fluorescence signal intensity value, and finally the position with the maximum fluorescence signal intensity value is accurately determined.

[0012] Optimally, the large step length is 50 microns, and the small step length is 10 microns.

[0013] Further, the average pixel intensity of the fluorescence image collected by the camera is calculated according to formula (1) in step S3, and the calculated average pixel intensity is taken as the fluorescence signal intensity value of the motorized displacement stage moving to the current position: (1). Wherein: represents the fluorescence signal intensity value of the motorized displacement stage moving to the current position, represents the intensity value of the th pixel grid, represents the background noise value of the th pixel grid, represents the total number of pixel grids.

[0014] Further, the computer automatically records each motorized displacement stage position 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 takes the peak point position as the optimal oblique layered light sheet illumination position.

[0015] A system for automatically finding an optimal oblique layered light sheet illumination position, for performing the method for automatically finding an optimal oblique layered light sheet illumination position according to any one of the preceding embodiments, comprising a laser, a laser collimation and expansion module, a first mirror, a motorized displacement stage, a dichroic mirror, a TIRF objective, a cover glass, a second mirror, a camera, and a computer; The laser is used to emit excitation light; The laser collimation and expansion module is used to collimate and expand the excitation light; The first mirror is used to adjust the excitation light incident to the motorized displacement stage; The motorized displacement stage is provided with a motorized displacement stage internal light path, for adjusting the position of the excitation light incident to the TIRF objective; The dichroic mirror is arranged at the intersection of the excitation light and the emission light, for reflecting the excitation light into the TIRF objective, and transmitting the emission light generated after the sample is excited to the second mirror; The TIRF objective is used to receive the excitation light, then converges the excitation light to form a focused layered light sheet, irradiates on the sample carried by the cover glass, and collects the emission light generated after the sample is excited, and shoots toward the dichroic mirror; The second mirror is used to reflect the emission light transmitted by the dichroic mirror into the camera; The camera is used to collect a fluorescence image; The computer is used to control the motorized displacement stage to move step by step according to a preset step size, and perform data processing.

[0016] Optimized, the laser collimation beam expander module comprises a first plano-convex lens and a second plano-convex lens, and the internal light path of the motorized displacement table comprises a third mirror, a fourth mirror and a third plano-convex lens, the excitation light reflected by the first mirror is vertically reflected to the third mirror and horizontally emitted to the third plano-convex lens after being reflected by the fourth mirror.

[0017] Advantages of the application: By connecting the computer with the motorized displacement table and the camera, the two are controlled to work cooperatively, the excitation light illumination position can be automatically adjusted, the best oblique lamellar light sheet illumination position can be quickly and accurately found, the error caused by artificial subjective judgment is avoided, the automation, self-adaptation, accuracy and other advantages are achieved, the quality of the reconstructed image of the single molecule positioning super-resolution imaging is significantly improved, and the system does not need to introduce additional devices, and the cost is relatively low. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a process schematic diagram of the application.

[0019] Figure 2a It is a comparison schematic diagram of the oblique lamellar light sheet illumination (HILO) and the traditional drop illumination (Epi) and total internal reflection illumination (TIRF) mode.

[0020] Figure 2b It is a light receiving schematic diagram of a sample under the oblique lamellar light sheet illumination (HILO).

[0021] Figure 3a It is a position-intensity relationship curve diagram obtained during coarse scanning of the application.

[0022] Figure 3b It is a position-intensity relationship curve diagram obtained during fine scanning of the application.

[0023] Figure 4 It is a system schematic diagram of the application.

[0024] In the figure: 1, laser; 2, first plano-convex lens; 3, second plano-convex lens; 4, first mirror; 5, third mirror; 6, fourth mirror; 7, third plano-convex lens; 8, motorized displacement table; 9, dichroic mirror; 10, TIRF objective lens; 11, cover glass; 12, sample; 13, second mirror; 14, camera; 15, computer. DETAILED DESCRIPTION

[0025] A method for automatically finding the best oblique lamellar light sheet illumination position, a process schematic diagram thereof is shown in Figure 1 The method comprises the following steps: S1: the excitation light emitted by the laser is collimated and expanded by the laser collimation and expansion module, is adjusted to enter the internal optical path of the electric displacement table by the first mirror, is adjusted to enter the dichroic mirror by the internal optical path of the electric displacement table, is reflected into the TIRF objective by the dichroic mirror, is converged to form a focused layered light sheet by the TIRF objective after the excitation light is converged by the TIRF objective, and is irradiated on the sample carried by the cover glass, the emission light generated by the sample after being excited is collected by the TIRF objective, and then the emission light is transmitted by the dichroic mirror and reflected by the second mirror to enter the camera; The laser collimation and expansion module can include a first plano-convex lens and a second plano-convex lens, which form an integral whole to collimate and expand the excitation light.

[0026] The internal optical path of the electric displacement table includes a third mirror, a fourth mirror and a third plano-convex lens, the excitation light reflected by the first mirror is vertically reflected to the third mirror and is horizontally reflected to the third plano-convex lens after being reflected by the fourth mirror. The third mirror, the fourth mirror and the third plano-convex lens are carried by the electric displacement table, and the position of the excitation light entering the TIRF objective can be adjusted by moving the electric displacement table.

[0027] The electric displacement table can be any programmable displacement table with high precision and high repeat positioning accuracy, and the step precision of the electric displacement table can be preferably 10 microns, and the repeat positioning accuracy can be preferably 1 micron.

[0028] The camera can be preferably an sCMOS camera suitable for single molecule localization super-resolution imaging. The sCMOS camera is the preferred camera applied to high-throughput fluorescence imaging, microscopic imaging, cold atom or quantum research, space debris monitoring and solar astronomy, etc. It has the characteristics of high resolution, ultra-low noise, large format, high performance, high sensitivity, low noise, high frame rate, etc., and is more suitable for single molecule localization super-resolution imaging.

[0029] S2: the computer controls the electric displacement table to move step by step according to a preset step size, and synchronously acquires the fluorescence images collected by the camera; Specifically, the computer can control the electric displacement table to move step by step according to the preset step size by the following method: first, control the electric displacement table to perform coarse scanning with a large step size, and position to the region of the maximum fluorescence signal intensity, then perform fine scanning with a small step size near the region of the maximum fluorescence signal intensity, until the position of the maximum fluorescence signal intensity is accurately determined.

[0030] Here, the large step size can be preferably 50 microns, and the small step size can be preferably 10 microns.

[0031] The computer controls the electric displacement table to move step by step along the optical path direction, at each set position, the excitation light irradiates the sample to make it emit fluorescence, and then the fluorescence signal is collected by the camera to generate a corresponding fluorescence image.

[0032] By first controlling the motorized displacement stage to perform coarse scanning with large step size, the region of maximum fluorescence signal intensity can be quickly located, and then fine scanning with small step size is performed near the region of maximum fluorescence signal intensity, which can further improve the speed of finding the position of maximum fluorescence signal intensity.

[0033] S3: Calculate the average pixel intensity of the selected region of the fluorescence image collected by the camera as the fluorescence signal intensity value of the motorized displacement stage moving to the current position, and determine the position point with the maximum fluorescence signal intensity value as the optimal oblique layered light sheet illumination position.

[0034] Specifically, the computer can calculate the average pixel intensity of the fluorescence image collected by the camera according to formula (1), and take the calculated average pixel intensity as the fluorescence signal intensity value of the motorized displacement stage moving to the current position: (1) ; Wherein: represents the fluorescence signal intensity value of the motorized displacement stage moving to the current position, represents the intensity value of the th pixel grid, represents the background noise value of the th pixel grid, represents the total number of pixel grids.

[0035] According to formula (1), the average pixel intensity of the selected region of the fluorescence image collected by the camera is calculated, and the calculated average pixel intensity is taken as the fluorescence signal intensity value of the motorized displacement stage moving to the current position, which can subtract the background noise from the original fluorescence image collected by the camera to obtain a net fluorescence signal image, so that the optimal oblique layered light sheet illumination position found is more accurate.

[0036] During the process of finding the optimal oblique layered light sheet illumination position, the computer can automatically record each motorized displacement stage position 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 peak point position as the optimal oblique layered light sheet illumination position.

[0037] This method realizes objective and accurate determination of the illumination position through automatic scanning and intensity quantification analysis, effectively avoiding subjective errors of manual adjustment.

[0038] The specific oblique layered light sheet illumination (HILO) and the comparison diagram of the traditional epi-illumination (Epi) and total internal reflection fluorescence illumination (TIRF) are shown in Figure 2a , and the light receiving schematic diagram of the sample under oblique layered light sheet illumination (HILO) is shown in Figure 2b . 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 mirror is used for reflecting the emitted light transmitted by the dichroic mirror into the camera. The camera is used for collecting the fluorescent image. The computer is used for controlling the electric displacement table to move step by step according to a preset step length and performing data processing.

[0041] Optimally, the laser collimation beam expander module comprises a first plano-convex lens 2 and a second plano-convex lens 3, and the internal optical path of the electric displacement table comprises a third mirror 5, a fourth mirror 6 and a third plano-convex lens 7, the excitation light reflected by the first mirror is reflected vertically to the third mirror and then horizontally out of the third plano-convex lens after being reflected by the fourth mirror.

[0042] In conclusion, the system and method for automatically finding the optimal oblique layered light sheet illumination position provided by the present application can automatically adjust the excitation light illumination position without introducing additional devices, thereby quickly and accurately finding the optimal oblique layered light sheet illumination position, avoiding the error of artificial subjective judgment, and having the advantages of automation, self-adaptation, high accuracy and the like, and significantly improving the reconstructed image quality of single molecule localization super-resolution imaging.

[0043] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for automatically finding the optimal illumination position of an inclined layered light sheet, characterized in that: 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.

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 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.

4. 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.

5. 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.

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

7. 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.

8. 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.

9. 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 8, 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 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.

10. A system for automatically finding the optimal illumination position of an inclined layered light sheet according to claim 9, characterized in that: 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 reflector, a fourth reflector, and a third plano-convex lens. The excitation light reflected by the first reflector is vertically reflected to the third reflector, and after being reflected by the fourth reflector, it is horizontally emitted to the third plano-convex lens.

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