A method for ghost correction in dual-channel quantitative FRET microscopy

Abstract

The present invention discloses a ghost correction method for dual-channel quantitative FRET microscopy, comprising the following steps: S1. Calculate the average offset of ghosts according to the dichroic mirror and optical path parameters used in the dual channels; S2. Collect images with a dual-channel microscope; S3. Determine whether the image is collected by the reflection channel; if not, output the image; if so, obtain the image data and proceed to step S4; S4. Plot the histogram of the image intensity distribution, determine the background value, find the left and right ghost positions for each pixel position respectively, and calculate the intensity ratios of the cell region to the left and right ghost regions; S5. Perform correction using the ghost correction algorithm; S6. Convert the corrected image to 16-bit depth and output the image. The method of the present invention performs ghost correction on a dual-channel wide-field fluorescence microscope with a single-sleeve lens, solves the ghost problem brought by the system, improves the imaging quality; at the same time, improves the accuracy of quantitative FRET analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical imaging, and particularly relates to a ghost correction method for dual-channel quantitative FRET microscopy imaging. Background Art

[0002] Biomacromolecules are the carriers of all life activities. How to monitor reversible dynamic molecular events in living cells has always been an important challenge in life science research. Fluorescence Resonance Energy Transfer (FRET) technology is currently the only imaging technology that can in-situ and real-time monitor weak and reversible dynamic molecular events in living cells.

[0003] Quantitative FRET measurements can be carried out under a confocal microscope or a wide-field fluorescence microscope. As an infinity optical system, a dual-channel wide-field fluorescence microscope requires a tube lens to converge the parallel light emitted from the objective lens onto the focal plane of the camera to acquire images. Because the optical path is relatively compact, the dual-channel microscope design with a single tube lens is adopted by many laboratories and is a commonly used technology. However, the dual-channel system with a single tube lens will have ghosts in the reflection channel during imaging, which not only affects the direct observation of cells by experimenters but also affects the accuracy of quantitative data analysis, especially in the three-channel quantitative FRET analysis. The main reasons are, firstly, the ghost causes the loss of fluorescence signals in the cell area, resulting in a decrease in cell brightness; secondly, the fluorescence emitted by different organelles in different regions of the cell is wrongly overlapped. Therefore, how to correct the ghosts in the reflection channel of the dual-channel microscope with a single tube lens is particularly important for accurate quantitative FRET analysis. Summary of the Invention

[0004] The main purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and propose a ghost correction method for dual-channel quantitative FRET microscopy imaging.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A ghost correction method for dual-channel quantitative FRET microscopy imaging, based on a dual-channel FRET quantitative microscopy imaging system, includes the following steps:

[0007] S1. Calculate the average offset of the ghost according to the dichroic mirror and optical path parameters used in the dual-channel;

[0008] S2. The dual-channel microscope acquires images;

[0009] S3. Determine whether the image is acquired from the reflection channel; if not, output the image; if so, obtain the image data and enter step S4;

[0010] S4. Draw a histogram of the image intensity distribution, determine the background value, find the left and right ghost positions for each pixel position respectively, and calculate the intensity ratios of the cell region to the left and right ghost regions.

[0011] S5. Perform correction using the ghost correction algorithm.

[0012] S6. Convert the corrected image to 16-bit depth and output the image.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] 1. The present invention performs ghost correction on a dual-channel wide-field fluorescence microscope with a single-sleeve lens, solves the ghost problem brought by the system, improves the imaging quality, and at the same time improves the accuracy of quantitative FRET analysis.

[0015] 2. The ghost correction algorithm of the present invention is a global correction for the image. The algorithm does not need to identify the cell contour and can effectively solve the correction of the ghost of cells overlapping in the Z-axis direction.

[0016] 3. The ghost correction algorithm of the present invention is applicable to various scenarios of dual-channel wide-field fluorescence microscopes with single-sleeve lenses, and only needs to simply change the parameters to adapt to dichroic mirrors with different thicknesses and different substrate materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a flowchart of the method of the present invention;

[0018] Figure 2 is a display diagram of the ghost image before and after correction;

[0019] Figure 3 is a processing flowchart of the dual-channel FRET quantitative microscopy imaging system in the embodiment;

[0020] Figure 4 is a system optical path diagram of the dual-channel FRET quantitative microscopy imaging system in the embodiment;

[0021] Figure 5 is a three-view drawing of the dual-channel FRET quantitative microscopy imaging system in the embodiment;

[0022] Figure 6 is a structural diagram of the dual-channel multi-dimensional adjustment bracket in the embodiment;

[0023] Figure 7 is a schematic diagram of the calculation result of d x in the embodiment;

[0024] Description of the attached reference numerals: 1 - Microscope bright-field illumination module; 2 - High-precision XY-axis motorized stage of the microscope; 3 - Microscope objective; 4 - First-stage beam splitting module of the microscope; 5 - High-precision Z-axis motorized displacement stage of the microscope; 6 - Dielectric mirror of the microscope; 7 - Dielectric mirror of the microscope; 8 - Mount for the sCMOS camera in the reflection channel of the microscope; 9 - Mount for the sCMOS camera in the direct-through channel of the microscope; 10 - Collimator of the microscope; 11 - sCMOS camera in the reflection channel of the microscope; 12 - Two-dimensional adjustment mount for the sCMOS camera in the reflection channel of the microscope; 13 - Two-dimensional adjustment mount for the sCMOS camera in the direct-through channel of the microscope; 14 - Second-stage beam splitting module of the microscope; 15 - sCMOS camera in the direct-through channel of the microscope; 16 - XYZ three-axis motor drive of the microscope; 17 - Dovetail groove for mounting the dual-camera module of the microscope. Detailed implementation manners

[0025] The present invention will be further described in detail below in conjunction with the embodiments and the attached drawings, but the implementation manners of the present invention are not limited thereto.

[0026] Embodiment

[0027] As Figure 1 shown, the present invention, a method for ghost correction in dual-channel quantitative FRET microscopy imaging, is based on a dual-channel FRET quantitative microscopy imaging system and includes the following steps:

[0028] S1. Calculate the average offset of the ghost according to the beam splitting dichroic mirror and the optical path parameters in the used dual channels; specifically:

[0029] According to the law of refraction, the distance of the outgoing light beam continuously reflected by the dichroic mirror on the camera target surface is d x , d x is expressed as:

[0030]

[0031] wherein, n1 is the refractive index of the medium where the incident light is located, n2 is the refractive index of the medium where the refracted light is located, θ is the incident angle of the light beam, and L is the thickness of the dichroic mirror;

[0032] In this embodiment, since a 1-inch sleeve is used in the microscope and the working distance of the sleeve lens used is 130 mm, the incident angle θ range of the second dichroic mirror is 39.4° to 50.6°, the thickness L of the dichroic mirror is 1 mm, the dichroic mirror substrate is made of fused silica with a refractive index of 1.45845; the refractive index of air is 1.00027; according to the d x formula, calculate with a step of 0.01° to obtain the average value of d x which is 0.7808 mm. AsFigure 7 As shown, it is for d x Schematic diagram of the calculation result of

[0033] S2. Collect images with a dual-channel microscope;

[0034] S3. Determine whether the image is collected by the reflection channel; if not, output the image; if so, obtain the image data and proceed to step S4; where, after obtaining the image data, it also includes:

[0035] Convert the two-dimensional array of grayscale image data detected by the camera into a double type for calculation.

[0036] S4. Draw a histogram of the image intensity distribution, determine the background value, find the left and right ghost positions for each pixel position respectively, and calculate the intensity ratios of the cell region to the left and right ghost regions; the method for determining the background value is specifically:

[0037] Statistically analyze the grayscale value distribution of the image data, with the abscissa being the frequency and the ordinate being the grayscale value. After pixel-by-pixel statistics, set the peak value of the first peak as the background value;

[0038] The method for finding the left and right ghost positions for each pixel position respectively is:

[0039] Each pixel corresponds to a coordinate in the two-dimensional array. According to the calculated Perform left and right coordinate conversion, set the left ghost as region A, and the right ghost as region B.

[0040] The method for calculating the intensity ratios of the cell region to the left and right ghost regions is specifically:

[0041] By experimental methods, statistically analyze the ghost images of different fluorescence samples, circle a region in the cell region, and define the average grayscale value of this region as I O ; According to Translate the region to find the left ghost region, and define the average grayscale value of the left ghost region as I A , find the right ghost region, and define the average grayscale value of the right ghost region as I B , and finally calculate according to the following formula:

[0042] t1 = I O / I A

[0043] t2 = I O / I B

[0044] Where, t1 represents the intensity ratio of the cell region to the left ghost region, and t2 represents the intensity ratio of the cell region to the right ghost region;

[0045] The average value of t1 and t2 obtained from different fluorescence sample images is calculated to obtain the final ghosting intensity ratio.

[0046] In this embodiment, through the method of experimental statistics, the ghosting intensity ratios of MCF-7 cells transfected with CFP, C4Y, C10Y, and C40Y were respectively statistically analyzed during imaging, and t1 = t2 = 6.6 was taken.

[0047] S5. Perform correction using the ghosting correction algorithm; the ghosting correction process is expressed as:

[0048] g(x, y) = T[f(x, y)]

[0049] Among them, g(x, y) represents the gray value of the pixel with coordinates (x, y) in the image after ghosting correction; f(x, y) represents the gray value of the pixel with coordinates (x, y) in the image; T represents the ghosting correction algorithm:

[0050]

[0051]

[0052]

[0053] Among them, M and N represent the number of horizontal and vertical pixels of an image with a size of M * N, and the unit is pixel.

[0054] S6. Convert the corrected image to 16-bit depth and output the image.

[0055] As Figure 2 shown, for MCF-7 cells expressing C40Y under an excitation light of 435 nm, with an excitation intensity of 80% and an exposure time of 800 ms, ghosting will occur in the reflection channel. In this embodiment, the thickness of the dichroic mirror 2 is 1 mm, and the base material used is fused silica. The working distance of the sleeve lens is 130 mm. After correction by the ghosting correction algorithm, the ghosting can be effectively corrected.

[0056] In this embodiment, the dual-channel FRET quantitative microscopy imaging system specifically includes a bright-field illumination module, a light source and collimation optical path module, a motorized stage displacement stage module, a fluorescence collection and focusing module, a double mirror group, a double beam splitting module, as well as a double sCMOS camera and an adjustment module;

[0057] The bright-field illumination module uses an LED white light bead as the bright-field light source of the microscope;

[0058] The light source and collimation optical path module uses a six-wavelength LED light source as the excitation light source for the fluorescence sample to generate a fluorescence signal on the sample surface;

[0059] The electric stage displacement stage module uses a high-precision electric XY stage to electrically switch the field of view of the fluorescent sample, and uses a high-precision electric Z-axis displacement stage to drive the objective lens for focusing.

[0060] The fluorescence collection and focusing module includes an objective lens and a sleeve lens. The objective lens is used to collect the fluorescence signal emitted by the fluorescent sample, and the sleeve lens focuses the parallel light emitted by the objective lens.

[0061] The double mirror group module includes a first dielectric film mirror and a second dielectric film mirror. The first dielectric film mirror reflects the parallel light in the collimated light path by 90°; the second dielectric film mirror reflects the parallel light emitted by the objective lens by 90° so that it enters the sleeve lens.

[0062] The double sCMOS and adjustment module specifically includes a first sCMOS camera and a second sCMOS camera, which are used as imaging devices for the donor detection channel and the acceptor detection channel in FRET quantitative analysis respectively.

[0063] The double beam splitting module includes a first beam splitting module and a second beam splitting module;

[0064] The first beam splitting module includes a first dichroic mirror and a first emission filter. The excitation light emitted from the collimated light path first passes through the first dichroic mirror and is reflected into the objective lens. The emission light of the fluorescent sample is transmitted through the first dichroic mirror and then transmitted through the first emission filter.

[0065] The second beam splitting module includes a second dichroic mirror, a second emission filter, and a third emission filter. The second beam splitting module separates the donor detection channel and the acceptor detection channel required for FRET quantitative analysis; the focused light emitted from the sleeve lens passes through the second dichroic mirror, and the second dichroic mirror selects different wavelengths in the focused light. The light of a part of the wavelengths is reflected by the second dichroic mirror and then reaches the first sCMOS camera after being filtered by the second emission filter. The light of the other part of the wavelengths is transmitted through the second dichroic mirror and then reaches the second sCMOS camera after being filtered by the third emission filter.

[0066] The light source and collimated light path module can directly incident the collimated excitation light into the first beam splitting module;

[0067] When the light is incident from the objective lens, the second dielectric film mirror can be cancelled, and the light is directly incident onto the sleeve lens and then enters the second beam splitting module.

[0068] The double sCMOS and adjustment module uses a two-dimensional adjustment bracket or a multi-dimensional adjustment bracket.

[0069] The first beam splitting module and the second beam splitting module can be manually or electrically switched by a turntable to adapt to different fluorescent dyes and FRET sample imaging systems;

[0070] The emission filters in the first beam splitting module and the second beam splitting module are used to eliminate unwanted optical signals, and the emission filters are switched using a manual or electric rotating wheel.

[0071] As Figure 5 shown, the three views of the dual-channel FRET quantitative microscopy system in this embodiment are presented. In this embodiment, the microscope bright-field illumination module 1 uses a 5W white light LED from CREE as the light source; the microscope high-precision XY-axis motorized stage 2 uses a stage from Jiancheng Optoelectronics with a 110*75mm travel and a closed-loop accuracy of 50nm; the microscope objective 3 uses a 60x apochromatic objective from Olympus; in the first stage beam splitting module 4 of the microscope, Chroma's ZT440 / 514 / 561 / 640rpc is used as the dichroic mirror, and the first emission filter uses Chroma's ET CFP / YFP / Cy5m; the microscope high-precision Z-axis motorized displacement stage 5 uses a 6mm travel displacement stage from Jiancheng Optoelectronics with a closed-loop accuracy of 10nm; the microscope dielectric mirror 6 uses a 1-inch dielectric mirror from Lubang; the microscope dielectric mirror 7 uses a 1-inch dielectric mirror from Lubang; the mounting bracket 8 for the microscope reflection channel sCMOS camera is a custom non-standard part; the mounting bracket 9 for the microscope direct-through channel sCMOS camera is a custom non-standard part; the microscope collimator lens 10 is a custom non-standard part; the microscope reflection channel sCMOS camera 11 uses a Hamamatsu Flash4.0V2 camera and is set on the two-dimensional adjustment bracket 12 for the microscope reflection channel sCMOS camera; the two-dimensional adjustment bracket 13 for the microscope direct-through channel sCMOS camera is a custom non-standard part; the second stage beam splitting module 14 of the microscope uses Chroma's T525lpxr-UF1 for beam splitting; the microscope direct-through channel sCMOS camera 15 uses a Hamamatsu Flash4.0V2 camera; the microscope XYZ three-axis motor driver 16 is a motor driver from Jiancheng Optoelectronics that is used in combination with the XY-axis stage and the Z-axis displacement stage; the dovetail groove 17 for mounting the microscope dual-camera module is a custom non-standard part.

[0072] As Figure 4 shown, the first dichroic mirror reflects light in the 435nm and 515nm bands and transmits light in the 460nm and 540nm bands. The emitted light from the fluorescent sample is collected by the objective lens and then converged using a sleeve lens. The second dichroic mirror reflects light in the 460nm band and transmits light in the 540nm band, which are respectively collected by the first sCMOS camera and the second sCMOS camera. Through such a combination method, rapid quantitative FRET analysis can be achieved for different excitation lights without switching filters and dichroic mirrors.

[0073] As Figure 6As shown, before performing quantitative FRET analysis, the images of the reflection channel and the direct-through channel are precisely registered using a dual-channel multi-dimensional adjustment mount.

[0074] As Figure 3 shown, quantitative FRET analysis is performed on the dual-channel FRET quantitative microscopy imaging system of this embodiment using the E-FRET method. Cells expressing CFP (cyan fluorescent protein) in MCF-7 cells are excited with light at 435 nm, with an excitation intensity of 20%, an exposure time of 500 ms, and a dual-sCMOS camera respectively acquires the reflection channel image and the direct-through channel image (select cells with a signal-to-noise ratio greater than 3), and the reflection channel image is corrected for ghosting; the crosstalk factors c and d are calculated;

[0075] Cells expressing YFP (yellow fluorescent protein) in MCF-7 cells are excited with light at 515 nm, with an excitation intensity of 20%, an exposure time of 500 ms, and a dual-sCMOS camera respectively acquires the reflection channel image and the direct-through channel image (select cells with a signal-to-noise ratio greater than 3), and the reflection channel image is corrected for ghosting; the crosstalk factors a and b are calculated;

[0076] Cells expressing C4Y, C10Y, and C40Y (all standard plasmids) in MCF-7 cells are respectively excited with light at 435 nm and 515 nm, with an excitation intensity of 20%, an exposure time of 500 ms, and a dual-sCMOS camera respectively acquires the reflection channel image and the direct-through channel image (select cells with a signal-to-noise ratio greater than 3), and the reflection channel image is corrected for ghosting; the system correction factors G, k, and E D and R C .

[0077] When performing quantitative FRET analysis using the E-FRET method, taking C40Y as an example, according to the literature report, its FRET efficiency should be 15.8%. If ghosting correction is not performed, the E D value obtained by E-FRET analysis is 14.0%, and the error rate is 11.4%; if ghosting correction is performed, the E D value obtained by E-FRET analysis is 15.4%, and the error rate is only 2.5%.

[0078] It should also be noted that in this specification, terms such as "including", "comprising" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "including one..." does not exclude the presence of additional identical elements in the process, method, article or device including said element.

[0079] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A ghost correction method for dual-channel quantitative FRET microscopy imaging, characterized in that, Based on a dual-channel FRET quantitative microscopy imaging system, it includes the following steps: S1. Calculate the average offset of ghost images according to the dichroic mirror and optical path parameters in the two channels used; S2. The dual-channel microscope acquires images; S3. Determine whether the image is acquired by the reflection channel; if not, output the image; if so, obtain the image data and enter step S4; S4. Plot the histogram of image intensity distribution, determine the background value, find the left and right ghost positions for each pixel position respectively, and calculate the intensity ratios of the cell region to the left and right ghost regions; S5. Perform correction using the ghost correction algorithm; S6. Convert the corrected image to 16-bit depth and output the image; In step S1, the calculation according to the dichroic mirror and parameters in the two channels used is specifically as follows: According to the law of refraction, the distance of the emerging light beam that is continuously reflected in the dichroic mirror on the camera target surface is d x , d x is expressed as: Where n1 is the refractive index of the medium where the incident light is located, n2 is the refractive index of the medium where the refracted light is located, θ is the incident angle of the light, and L is the thickness of the dichroic mirror; According to the sleeve lens and its optical path parameters used in the microscope, the range of the incident angle θ is obtained, and combined with the calculation formula of d x , calculate step by step with a preset angle to obtain d x 's average value In step S3, after obtaining the image data, it further includes: Convert the two-dimensional array of grayscale image data detected by the camera into double type for operation; In step S4, the method for determining the background value is specifically as follows: Statistically analyze the grayscale value distribution of the image data, with the abscissa being the frequency and the ordinate being the grayscale value. After pixel-by-pixel statistics, set the peak value of the first peak as the background value; The method for finding the left and right ghost positions for each pixel position respectively is: Each pixel corresponds to a coordinate in a two-dimensional array. According to the calculated perform the conversion of the left and right coordinates. Set the left ghosting as area A and the right ghosting as area B; Calculate the intensity ratios of the cell region to the left and right ghost regions, specifically as follows: By experimental methods, count the ghost images of different fluorescent samples, circle a region in the cell area, and define the average gray value of this region as I O ; According to Translate the region to find the left ghost region, and define the average gray value of the left ghost region as I A , find the right ghost region, and define the average gray value of the right ghost region as I B , and finally calculate according to the following formula: t1 = I O / I A t2 = I O / I B Where t1 represents the intensity ratio of the cell region to the left ghost region, and t2 represents the intensity ratio of the cell region to the right ghost region; Based on t1 and t2 obtained from different fluorescence sample pictures, calculate the average value to obtain the final ghost intensity ratio; In step S5, the ghost correction process is expressed as: g(x,y) = T[f(x,y)] Where g(x,y) represents the grayscale value of the pixel with coordinates (x,y) in the image after ghost correction; f(x,y) represents the grayscale value of the pixel with coordinates (x,y) in the image; T represents the ghost correction algorithm: Where M and N represent the number of horizontal and vertical pixels of an image with a size of M*N, in pixels.

2. A ghost correction method for dual-channel quantitative FRET microscopy according to claim 1, characterized in that The dual-channel FRET quantitative microscopy imaging system specifically includes a bright-field illumination module, a light source and collimation optical path module, an electric stage displacement stage module, a fluorescence collection and focusing module, a double mirror group, a double beam splitting module, as well as a double sCMOS camera and an adjustment module; The bright-field illumination module serves as the bright-field light source of the microscope; The light source and collimation optical path module serves as the excitation light source for the fluorescence sample, generating a fluorescence signal on the surface of the sample; The electric stage displacement stage module uses an electric XY-axis stage to electrically switch the field of view of the fluorescence sample, and uses an electric Z-axis displacement stage to drive the objective lens for focusing; The fluorescence collection and focusing module includes an objective lens and a sleeve lens. The objective lens is used to collect the fluorescence signal emitted by the fluorescence sample, and the sleeve lens focuses the parallel light emitted by the objective lens; Dual mirror group module, including a first mirror and a second mirror. The first mirror reflects the parallel light in the collimated light path by 90°. The second mirror reflects the parallel light emitted by the objective lens by 90° to make it enter the sleeve lens.

3. A ghost correction method for dual-channel quantitative FRET microscopy according to claim 2, characterized in that, Dual sCMOS and adjustment module, specifically including a first sCMOS camera and a second sCMOS camera, which are respectively used as imaging devices for the donor detection channel and the acceptor detection channel in FRET quantitative analysis.

4. A ghost correction method for dual-channel quantitative FRET microscopy according to claim 3, characterized in that, Dual beam splitting module, including a first beam splitting module and a second beam splitting module; The first beam splitting module includes a first dichroic mirror and a first emission filter. The excitation light emitted from the collimated light path first passes through the first dichroic mirror and is reflected into the objective lens. The emission light of the fluorescent sample transmits through the first dichroic mirror and then transmits through the first emission filter; The second beam splitting module includes a second dichroic mirror, a second emission filter, and a third emission filter. The second beam splitting module separates the donor detection channel and the acceptor detection channel required for FRET quantitative analysis. The focused light emitted from the sleeve lens passes through the second dichroic mirror. The second dichroic mirror selects different wavelengths in the focused light. The light of a part of the wavelengths is reflected by the second dichroic mirror and then reaches the first sCMOS camera after being filtered by the second emission filter. The light of the other part of the wavelengths is transmitted by the second dichroic mirror and then reaches the second sCMOS camera after being filtered by the third emission filter.

5. A ghost correction method for dual-channel quantitative FRET microscopy according to claim 4, characterized in that, Light source and collimated light path module, which can directly incident the collimated excitation light into the first beam splitting module; When the light is incident from the objective lens, the second mirror can be removed, and the light can be directly incident on the sleeve lens and then enter the second beam splitting module.

6. A ghost correction method for dual-channel quantitative FRET microscopy imaging according to claim 1, characterized in that, The dual sCMOS and adjustment module uses a two-dimensional adjustment bracket or a multi-dimensional adjustment bracket.

7. A ghost correction method for dual-channel quantitative FRET microscopy imaging according to claim 1, characterized in that, The first beam splitting module and the second beam splitting module can be manually or electrically switched by a turntable to adapt to different fluorescent dyes and FRET sample imaging systems; The emission filters in the first beam splitting module and the second beam splitting module are used to eliminate unnecessary optical signals, and the emission filters are switched by a manual or electric rotating wheel.

Images