Multicolor fluorescence fluctuation imaging system and method based on a slowly varying transmission ratio dichroic mirror

Abstract

The present application relates to a multi-color fluorescence fluctuation imaging system and method based on a slowly varying transmittance dichroic mirror, which comprises: using multiple wavelength quantum dot fluorescence to mark different subcellular organelle structures, and using a single wavelength excitation light to obtain multi-color fluorescence signals; using a slowly varying transmittance dichroic mirror to split the multi-color fluorescence signals, and using a single camera to simultaneously collect multi-color fluorescence signals in the reflection channel and the transmission channel; using a method based on high-order correlation function calculation to separate different wavelength fluorescence signals; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using a multi-wavelength channel signal mutual iteration method to reduce cross-color between different channels; using

Description

Technical Field

[0001] This invention relates to the technical field of optical imaging systems and synchronous multicolor fluorescence separation methods, particularly to a multicolor fluorescence fluctuation imaging system and method based on a dichroic mirror with gradually varying transmittance. Background Technology

[0002] Fluorescence fluctuation super-resolution technology utilizes the fluctuation characteristics of fluorescence signals to overcome the diffraction limit of traditional optical microscopy. Existing fluorescence fluctuation-based super-resolution microscopy techniques, such as Super-Resolution Optical Scintillation Microscopy (SOFI), Super-Resolution Radial Fluctuation Algorithm (SRRF), Fluorescence Fluctuation-Based Multi-Signal Classification Algorithm (MUSICAL), and Mean Shift Super-Resolution Algorithm (MSSR), can significantly improve imaging resolution. Current multicolor fluorescence microscopy primarily acquires multicolor images through spatial or temporal wavelength division. Spatially dividing the wavelength involves introducing multiple wavelength channels and image detection optical paths, which significantly increases the loss of multicolor fluorescence signals, increases system cost and complexity, and reduces system stability. Temporally dividing the wavelength involves acquiring fluorescence at different wavelengths from multicolor samples multiple times at different time periods, leading to time differences between wavelengths and significantly reducing the temporal resolution of the imaging. Techniques that separate multicolor spectra through multi-wavelength excitation and higher-order cumulant functions require multiple excitation optical paths, significantly increasing system complexity and reducing system stability. Furthermore, higher-order cumulant function analysis methods can introduce various artifacts that affect the quality of image reconstruction. Existing multicolor super-resolution imaging techniques typically achieve signal separation of multiple wavelengths during image sequence processing through spatial wavelength division acquisition or temporal wavelength division. Spatial wavelength division acquisition significantly increases the complexity and cost of the imaging system, while temporal wavelength division reduces the imaging rate. Therefore, further development of multicolor fluorescence microscopy is hampered by the trade-offs between the number of excitation optical paths, the number of occupied spatial wavelength channels, and the elimination of channel delays. This limits the application of fluorescence fluctuation-based super-resolution microscopy in life science research, such as the simultaneous observation of multicolor labeled subcellular organelles. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a multicolor fluorescence fluctuation imaging system and method based on a dichroic mirror with gradually varying transmittance. This method can realize the acquisition and separation of multicolor fluorescence samples using only a single wavelength excitation light, two fluorescence signal acquisition channels and a single camera image acquisition, effectively solving the problem of mutual constraints between the number of excitation light paths, the number of wavelength channels occupied and the elimination of channel delay.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a multicolor fluorescence fluctuation imaging system based on a dichroic mirror with gradually varying transmittance, comprising an excitation light source for providing an excitation beam;

[0005] A first converging lens focuses the excitation light from the excitation source, and the excitation light from the excitation source passes through the first converging lens;

[0006] A first dichroic mirror reflects the excitation light from the first converging lens;

[0007] An objective lens that transmits the excitation light from the first dichroic mirror;

[0008] A stage on which a biological cell sample is placed, and an excitation light from the objective lens irradiates the biological sample. The biological sample emits fluorescence when excited by the excitation light from the objective lens. The fluorescence is emitted through the objective lens to a first dichroic mirror, and the first dichroic mirror transmits the fluorescence from the objective lens.

[0009] A second converging lens focuses the fluorescence from the first dichroic mirror, and the fluorescence from the second dichroic mirror passes through the second converging lens;

[0010] A third converging lens focuses the fluorescence from the second converging lens, and the fluorescence from the second converging lens passes through the third converging lens;

[0011] A second dichroic mirror splits the fluorescence from the third converging lens. A portion of the fluorescence from the third converging lens is reflected by the second dichroic mirror, and another portion of the fluorescence from the third converging lens is transmitted through the second dichroic mirror.

[0012] A first reflecting mirror reflects the fluorescence that has been reflected by the second dichroic mirror;

[0013] A second mirror reflects the fluorescence that was reflected by the first mirror;

[0014] A third mirror reflects the fluorescence that was reflected by the second mirror;

[0015] A fourth reflecting mirror reflects the fluorescence transmitted by the second dichroic mirror;

[0016] A fifth reflecting mirror reflects the fluorescence that was reflected by the fourth reflecting mirror;

[0017] A sixth reflecting mirror reflects the fluorescence that was reflected by the fifth reflecting mirror;

[0018] A seventh reflecting mirror reflects the fluorescence reflected by the sixth reflecting mirror;

[0019] A CMOS camera is used to image the fluorescence reflected from the third mirror and to image the fluorescence reflected from the seventh mirror.

[0020] This invention also provides a multicolor fluorescence fluctuation imaging method based on a slowly varying transmittance dichroic mirror, employing the aforementioned multicolor fluorescence fluctuation imaging system based on a slowly varying transmittance dichroic mirror, comprising the following steps:

[0021] Step 1: Set up a super-resolution imaging system for synchronous multicolor fluorescence fluctuations based on a dichroic mirror with gradually varying transmittance. The super-resolution imaging system for synchronous multicolor fluorescence fluctuations based on a dichroic mirror with gradually varying transmittance includes an excitation source, a first converging lens, a first dichroic mirror, an objective lens, a stage, a second converging lens, a third converging lens, a second dichroic mirror with gradually varying transmittance, a first reflecting mirror, a second reflecting mirror, a third reflecting mirror, a fourth reflecting mirror, a fifth reflecting mirror, a sixth reflecting mirror, a seventh reflecting mirror, and a CMOS camera.

[0022] Step 2: Use six quantum dots with different fluorescence emission wavelengths to label different cell structures in biological samples, and use single-wavelength excitation light to excite the six quantum dots to emit light in a short wavelength range;

[0023] Step 3: Place and fix the labeled biological sample on the stage;

[0024] Step 4: Adjust the distance between the objective lens and the biological sample so that the biological sample is located at the focal point of the objective lens, so that the imaging system satisfies the object-image conjugate relationship of the optical microscopy system;

[0025] Step 5: The excitation light emitted by the excitation source is emitted through the first converging lens to the first dichroic mirror. The light reflected by the first dichroic mirror is emitted through the objective lens to illuminate the biological sample. The biological sample is excited and produces fluorescence. The fluorescence is emitted through the objective lens to the first dichroic mirror. The light transmitted through the first dichroic mirror is emitted to the second converging lens for convergence. The light transmitted through the second converging lens is emitted to the third converging lens for convergence. The light transmitted through the third converging lens is emitted to the second dichroic mirror with a gradually varying transmittance. The light reflected by the second dichroic mirror is reflected by the first reflecting mirror to the second reflecting mirror. The light reflected by the second reflecting mirror is reflected by the third reflecting mirror to the CMOS camera for fluorescence imaging. The light transmitted through the second dichroic mirror is emitted to the fourth reflecting mirror. The light reflected by the fourth reflecting mirror is reflected to the fifth reflecting mirror. The light reflected by the fifth reflecting mirror is reflected to the sixth reflecting mirror. The light reflected by the sixth reflecting mirror is reflected to the seventh reflecting mirror. The light reflected by the seventh reflecting mirror is reflected to the CMOS camera for fluorescence imaging.

[0026] Step 6: Use and adjust the output power of the single-wavelength excitation light to simultaneously excite quantum dots of multiple wavelengths to emit light, and observe and collect fluorescence images with a single camera. When obvious fluorescence intensity flickering is observed, stabilize the output power of the excitation light.

[0027] Step 7: Use a single camera to synchronously acquire the reflection and transmission channel signals of the dichroic mirror with gradually varying transmittance via the Sigmoid curve, and use a sub-pixel localization algorithm to locate the same area in the time series of the acquired reflection and transmission channels.

[0028] Step 8: Calculate the correlation function of different combinations of the two channel image sequences after localization by the subpixel localization algorithm, and form a system of equations to solve the corresponding monochromatic correlation function to separate the signals of different wavelengths;

[0029] Step 9: Use a multi-channel iterative method to reduce crosstalk in the separated monochromatic wavelength signals;

[0030] Step 10: Use the fluorescence images of different wavelengths after reducing crosstalk to perform super-resolution algorithm processing to obtain high-resolution images of different cell structures.

[0031] In a preferred embodiment, the first dichroic mirror is selected to reflect the excitation light from the first converging lens and transmit the fluorescence emitted by the six marked quantum dots at wavelengths that are also ...

[0032] In a preferred embodiment, the gradually varying transmittance is represented by an expression for the Sigmoid function:

[0033]

[0034] In the formula: λ is the wavelength; a and b are the slope parameters of the curve, with a taking the value of 0.05 and b taking the value of 600.

[0035] In a preferred embodiment, multiple sets of n-order correlation functions are obtained by combining different methods on the sub-pixel aligned dual-channel fluorescence signal image; the fluorescence signals generated by the reflection and transmission of the dichroic mirror 3-splitting based on the sigmoid function's slowly varying transmittance are represented as follows:

[0036]

[0037]

[0038] In the formula: r represents the pixel coordinates; t represents the signal acquisition time; S A Indicates the reflected channel signal and S B Indicates the transmission channel signal; A i B represents the reflectivity of the dichroic mirror for the i-th wavelength signal; i The transmittance and reflectance A of the dichroic mirror for the i-th wavelength signal are represented by A. i With transmittance B i The theoretical value of the sum is 1;

[0039] Calculate the nth-order correlation function Corr for the fluorescence fluctuation signals of the two channels. n ,get:

[0040]

[0041] In the formula: r represents the pixel coordinates; t represents the signal acquisition time; S A Indicates the reflected channel signal and S B Indicates the transmission channel signal; N is the number of wavelength channels; A i B represents the reflectivity of the dichroic mirror for the i-th wavelength signal; i S represents the transmittance of the dichroic mirror for the i-th wavelength signal; i (r,t) represents the intensity signal distribution of the i-th fluorescent molecule; Corr n {S i (r,t)} represents the monochromatic correlation function of the fluorescence fluctuation signal at wavelength i; calculating the nth-order correlation function by combining the reflected and transmitted channel signals yields n+1 equations; when the number of wavelength channels N equals n+1, the number of unknowns in the system of equations is equal to the number of equations, and the monochromatic correlation function Corr can be directly solved by solving the equations. n {S i (r,t)}, that is, extracting the signal for each wavelength;

[0042] The separated images at different wavelengths are processed to reduce crosstalk, resulting in individual images of each wavelength of the multicolor biological sample. The iterative formula for reducing crosstalk is shown below:

[0043]

[0044] In the formula: λ is the color mixing factor; λ (0 < λ < 1) is the scaling factor.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. This invention enables simultaneous fluorescent labeling of different cell structures in the same biological sample. It also proposes the use of a dichroic mirror with a slowly varying transmittance distribution curve constructed based on the Sigmoid function to separate multicolor fluorescence signals. Furthermore, it utilizes a synchronous multicolor fluorescence fluctuation super-resolution imaging algorithm to process the image sequence, thereby achieving the separation of signal light at different wavelengths. This solves the problem of mutual constraints between the number of excitation optical paths, the number of wavelength channels occupied, and the elimination of channel delay.

[0047] 2. The six quantum dots with different fluorescence emission wavelengths used in this invention are used as biomarkers, which can be excited by the same wavelength light source, greatly reducing the complexity and cost of the system.

[0048] 3. This invention uses two sets of mirrors to converge the fluorescence signals from the reflection and transmission channels, requiring only a single CMOS camera to acquire dual-channel multicolor fluorescence signals, significantly reducing the cost of imaging equipment. This invention can be widely applied in the fluorescence imaging of biological samples. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the structure of a synchronous multicolor fluorescence fluctuation super-resolution imaging system based on a dichroic mirror with gradually varying transmittance in a preferred embodiment of the present invention.

[0050] Figure 2 A schematic curve of transmittance of a gradually varying dichroic mirror based on a sigmoid curve is shown in a preferred embodiment of the present invention.

[0051] Figure 3 A schematic diagram of multicolor labeling of biological cell samples in a preferred embodiment of the present invention. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0053] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0054] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application; as used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise; furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0055] like Figure 1As shown, the present invention proposes a synchronous multicolor fluorescence fluctuation super-resolution imaging system based on a dichroic mirror with gradually varying transmittance. It includes an excitation light source 1, two dichroic mirrors 2 and 3, an objective lens 4, a stage 5, three converging lenses 6, 7 and 8, seven reflecting mirrors 9, 10, 11, 12, 13, 14 and 15, and a CMOS camera 16.

[0056] Excitation source 1 provides a single-wavelength excitation light. Converging lens 6 is positioned in the optical path of the excitation light and focuses the excitation light emitted from excitation source 1. Dichroic mirror 2 reflects the excitation light from converging lens 6, and the reflected excitation light shines onto the biological sample 17 on stage 5 through objective lens 4. The biological sample 17 emits fluorescence under excitation, and the fluorescence is emitted through objective lens 4 to dichroic mirror 2. Dichroic mirror 2 transmits the fluorescence from objective lens 4. Converging lens 7 is positioned in the optical path of the transmitted fluorescence from dichroic mirror 2 and focuses the fluorescence from dichroic mirror 2. Converging lens 8 is positioned in the optical path of the transmitted fluorescence from converging lens 7 and focuses the fluorescence from converging lens 7. Dichroic mirror 3 is positioned in the optical path of the transmitted fluorescence from converging lens 8 and disperses the fluorescence from converging lens 8; part of the fluorescence is reflected by dichroic mirror 3, and part of the fluorescence is transmitted by dichroic mirror 3. Reflector 9 is positioned in the optical path of the reflected fluorescence from dichroic mirror 3 and reflects the reflected fluorescence from dichroic mirror 3. Reflector 10 is positioned in the optical path of the reflected fluorescence from reflector 9 and reflects the reflected fluorescence from reflector 9. Reflector 11 is positioned in the optical path of the reflected fluorescence from reflector 10 and reflects the reflected fluorescence from reflector 10, thus imaged on the CMOS camera 16. Reflector 12 is positioned in the optical path of the transmitted fluorescence from dichroic mirror 3 and reflects the transmitted fluorescence from dichroic mirror 3. Reflector 13 is positioned in the optical path of the reflected fluorescence from reflector 12 and reflects the reflected fluorescence from reflector 12. Reflector 14 is positioned in the optical path of the reflected fluorescence from reflector 13 and reflects the reflected fluorescence from reflector 13. Reflector 15 is positioned in the optical path of the reflected fluorescence from reflector 14 and reflects the reflected fluorescence from reflector 14, thus imaged on the CMOS camera 16.

[0057] This invention uses a CMOS camera to acquire two signals—the transmission channel and the reflection channel—after spectral dispersion using a dichroic mirror based on slowly varying transmittance, to image sample 17 using a multi-frame fluorescence fluctuation image sequence. The acquired signals from the transmission and reflection channels are then sub-pixel aligned, and signals of different wavelengths are separated to obtain image sequences of fluorescence signals at different wavelengths. Each separated wavelength image is processed using a super-resolution algorithm, ultimately yielding high-resolution images of different cellular structures labeled with each quantum dot.

[0058] In the above embodiments, the excitation light source 1 can be a laser.

[0059] In the above embodiments, the objective lens 4 can be an objective lens with a numerical aperture greater than 1.4 so that the CMOS camera 16 can acquire wide-field images with the highest possible resolution, which facilitates the use of super-resolution fluctuation microscopy imaging algorithms to further improve the spatial resolution and break through the optical diffraction limit.

[0060] In the above embodiments, an inverted fluorescence microscope is used to image the biological sample 17. An upright fluorescence microscope can also be used; the only difference is that the light path is flipped. That is, when using an upright fluorescence microscope, the biological sample 17 is positioned at the lower part of the objective lens 4; when using an inverted fluorescence microscope, the biological sample 17 is positioned at the upper part of the objective lens 4 (e.g., ...). Figure 1 (As shown).

[0061] Based on the aforementioned synchronous multicolor fluorescence fluctuation super-resolution imaging system using a slowly varying transmittance dichroic mirror, this invention also proposes a synchronous multicolor fluorescence fluctuation super-resolution imaging method based on a slowly varying transmittance dichroic mirror, which includes the following steps:

[0062] 1) Quantum dots 18, 19, 20, 21, 22, and 23 with different fluorescence emission wavelengths were used to label different cell structures 24, 25, 26, 27, 28, and 29 in biological sample 17.

[0063] 2) Place and fix the labeled biological sample 17 on the stage 5.

[0064] 3) Finely adjust the distance between the objective lens 4 and the biological sample 17 so that the biological sample 17 is located at the focal point of the objective lens 4, so that the imaging system satisfies the object-image conjugate relationship of the optical microscopy system.

[0065] 4) Turn on the excitation light source 1, adjust the output power of the excitation light, and simultaneously observe the acquired fluorescence image through the CMOS camera 16. When obvious fluorescence intensity flickering is observed, stabilize the output power of the excitation light.

[0066] 5) Set an appropriate exposure time for the CMOS camera (usually 10–30 milliseconds), and simultaneously acquire data using the CMOS camera 16 based on slowly varying (e.g., ...) Figure 2 (As shown) The fluorescence signals reflected and transmitted by the dichroic mirror 3 (within 1000 frames) are collected. The slowly varying transmittance can be expressed by the Sigmoid function:

[0067]

[0068] In the formula: λ is the wavelength; a and b are the slope parameters of the curve, with a taking the value of 0.05 and b taking the value of 600.

[0069] 6) Use the subpixel localization algorithm to locate the same region for the time series images of the two acquired channels respectively.

[0070] 7) Calculate multiple sets of nth-order correlation functions by performing different combinations on the subpixel-aligned dual-channel fluorescence signal image. Theoretically, the fluorescence signal generated by the reflection and transmission of light through a dichroic mirror with slowly varying transmittance based on the Sigmoid function can be expressed as:

[0071]

[0072]

[0073] In the formula: r represents the pixel coordinates; t represents the signal acquisition time; S A Indicates the reflected channel signal and S B Indicates the transmission channel signal; A i B represents the reflectivity of the dichroic mirror for the i-th wavelength signal; i The transmittance and reflectance A of the dichroic mirror for the i-th wavelength signal are represented by A. i With transmittance B i The theoretical value of the sum is 1.

[0074] Calculate the nth-order correlation function Corr for the fluorescence fluctuation signals of the two channels. n We can obtain:

[0075]

[0076] In the formula: r represents the pixel coordinates; t represents the signal acquisition time; S A Indicates the reflected channel signal and S B Indicates the transmission channel signal; N is the number of wavelength channels; A i B represents the reflectivity of the dichroic mirror for the i-th wavelength signal; i S represents the transmittance of the dichroic mirror for the i-th wavelength signal; i (r,t) represents the intensity signal distribution of the i-th fluorescent molecule; Corr n {S i (r,t)} represents the monochromatic correlation function of the fluorescence fluctuation signal at wavelength i. Calculating the nth-order correlation function by combining the reflection and transmission channel signals yields n+1 equations. When the number of wavelength channels N equals n+1, the number of unknowns in the system of equations equals the number of equations, and the monochromatic correlation function Corr can be directly solved by solving these equations. n {S i (r,t)}, that is, extracting the signal for each wavelength.

[0077] 8) By performing color crosstalk reduction processing on the separated images of different wavelengths, individual images of each wavelength of the multicolor biological sample can be obtained. The iterative formula for color crosstalk reduction is shown below:

[0078]

[0079] In the formula: λ is the color mixing factor; λ (0 < λ < 1) is the scaling factor.

[0080] 9) Use the super-resolution algorithm SRRF or MSSR to process the image at each wavelength to achieve super-resolution fluorescence imaging of multicolor biological samples.

[0081] In the above embodiments, as Figure 3 As shown, biological sample 17 was labeled with six quantum dots 18, 19, 20, 21, 22, and 23. These six quantum dots 18, 19, 20, 21, 22, and 23 were labeled on different cell structures 24, 25, 26, 27, 28, and 29. Among them, the fluorescence emission center wavelength of quantum dot 18 is 525 nm, that of quantum dot 19 is 545 nm, that of quantum dot 20 is 565 nm, that of quantum dot 21 is 585 nm, that of quantum dot 22 is 625 nm, and that of quantum dot 23 is 655 nm. All six quantum dots can be excited by the same wavelength of excitation light.

[0082] In the above embodiments, the dichroic mirror 2 should select an appropriate wavelength for the different quantum dots being labeled. The dichroic mirror 2 should reflect the excitation light from the converging lens 6 and transmit the fluorescence emitted by the three labeled quantum dots 18, 19, 20, 21, 22, and 23; the dichroic mirror 3, based on the slowly varying transmittance of the Sigmoid curve, has different reflection and transmission coefficients for fluorescence signals of different wavelengths; the CMOS camera 16 acquires the signals from the reflection and transmission channels, and then uses a separation algorithm to separate the signals of different wavelengths.

[0083] This product is based on a wide-field fluorescence microscope. It only requires the construction of a spectral path based on a dichroic mirror with gradually varying transmittance. It excites multicolor fluorescence signals with a single wavelength and collects multicolor signals through dual channels of reflection and transmission. It does not require more channels and can use a single CMOS camera to achieve synchronous image acquisition of multicolor wavelength channels, providing a new method and approach for the study of synchronous multicolor observation of various subcellular structures.

[0084] A super-resolution imaging system and method for simultaneous multicolor fluorescence fluctuations based on a slowly varying transmittance dichroic mirror is disclosed. First, a single-wavelength excitation light excites quantum dots of multiple wavelengths to simultaneously emit fluorescence signals. Second, an imaging system based on a slowly varying transmittance dichroic mirror is used to split the multicolor fluorescence signals into reflection and transmission channels, and a single CMOS camera simultaneously acquires the dual-channel fluorescence signals. Next, a high-order correlation function based on fluorescence fluctuations is used to separate the reflection and transmission channel signals, achieving multicolor fluorescence signal separation. Then, crosstalk reduction processing is performed on each separated monochromatic fluorescence signal. Finally, a super-resolution algorithm is used to process the monochromatic fluorescence signals, achieving super-resolution imaging of multicolor biological samples.

[0085] The above embodiments are only used to illustrate the present invention. Any equivalent transformations and improvements made on the basis of the technical solutions of the present invention should not be excluded from the protection scope of the present invention.

Claims

1. A multicolor fluorescence fluctuation imaging system based on a dichroic mirror with gradually varying transmittance, characterized in that... Includes an excitation source for providing an excitation beam; A first converging lens focuses the excitation light from the excitation source, and the excitation light from the excitation source passes through the first converging lens; A first dichroic mirror reflects the excitation light from the first converging lens; An objective lens that transmits the excitation light from the first dichroic mirror; A stage on which a biological cell sample is placed, and an excitation light from the objective lens irradiates the biological cell sample. The biological cell sample emits fluorescence under the excitation light from the objective lens. The fluorescence is emitted through the objective lens to a first dichroic mirror, and the first dichroic mirror transmits the fluorescence from the objective lens. A second converging lens focuses the fluorescence from the first dichroic mirror, and the fluorescence from the first dichroic mirror passes through the second converging lens; A third converging lens focuses the fluorescence from the second converging lens, and the fluorescence from the second converging lens passes through the third converging lens; A second dichroic mirror splits the fluorescence from the third converging lens. A portion of the fluorescence from the third converging lens is reflected by the second dichroic mirror, and another portion of the fluorescence from the third converging lens is transmitted through the second dichroic mirror. A first reflecting mirror reflects the fluorescence that has been reflected by the second dichroic mirror; A second mirror reflects the fluorescence that was reflected by the first mirror; A third mirror reflects the fluorescence that was reflected by the second mirror; A fourth reflecting mirror reflects the fluorescence transmitted by the second dichroic mirror; A fifth reflecting mirror reflects the fluorescence that was reflected by the fourth reflecting mirror; A sixth reflecting mirror reflects the fluorescence that was reflected by the fifth reflecting mirror; A seventh reflecting mirror reflects the fluorescence reflected by the sixth reflecting mirror; A CMOS camera is used to image the fluorescence reflected from the third mirror and to image the fluorescence reflected from the seventh mirror. The imaging method includes the following steps: Step 1: Set up a super-resolution imaging system for synchronous multicolor fluorescence fluctuations based on a dichroic mirror with gradually varying transmittance. The super-resolution imaging system for synchronous multicolor fluorescence fluctuations based on a dichroic mirror with gradually varying transmittance includes an excitation source, a first converging lens, a first dichroic mirror, an objective lens, a stage, a second converging lens, a third converging lens, a second dichroic mirror with gradually varying transmittance, a first reflecting mirror, a second reflecting mirror, a third reflecting mirror, a fourth reflecting mirror, a fifth reflecting mirror, a sixth reflecting mirror, a seventh reflecting mirror, and a CMOS camera. Step 2: Use six quantum dots with different fluorescence emission wavelengths to label different cell structures in biological cell samples, and use single-wavelength excitation light to excite the six quantum dots to emit light in a short wavelength range; Step 3: Place and fix the labeled biological cell sample on the stage; Step 4: Adjust the distance between the objective lens and the biological cell sample so that the biological cell sample is located at the focal point of the objective lens, so that the imaging system satisfies the object-image conjugate relationship of the optical microscopy system; Step 5: The excitation light emitted by the excitation source is emitted through the first converging lens to the first dichroic mirror. The light reflected by the first dichroic mirror is emitted through the objective lens to irradiate the biological cell sample. The biological cell sample is excited and produces fluorescence. The fluorescence is emitted through the objective lens to the first dichroic mirror. The light transmitted through the first dichroic mirror is emitted to the second converging lens for convergence. The light transmitted through the second converging lens is emitted to the third converging lens for convergence. The light transmitted through the third converging lens is emitted to the second dichroic mirror. The light reflected by the second dichroic mirror is reflected by the first reflecting mirror to the second reflecting mirror. The light reflected by the second reflecting mirror is reflected by the third reflecting mirror to the CMOS camera for fluorescence imaging. The light transmitted through the second dichroic mirror is emitted to the fourth reflecting mirror. The light reflected by the fourth reflecting mirror is reflected to the fifth reflecting mirror. The light reflected by the fifth reflecting mirror is reflected to the sixth reflecting mirror. The light reflected by the sixth reflecting mirror is reflected to the seventh reflecting mirror. The light reflected by the seventh reflecting mirror is reflected to the CMOS camera for fluorescence imaging. Step 6: Use and adjust the output power of the single-wavelength excitation light to simultaneously excite quantum dots of multiple wavelengths to emit light, and observe and collect fluorescence images with a single camera. When obvious fluorescence intensity flickering is observed, stabilize the output power of the excitation light. Step 7: Use a single camera to synchronously acquire the reflection and transmission channel signals of the dichroic mirror with gradually varying transmittance via the Sigmoid curve, and use a sub-pixel localization algorithm to locate the same area in the time series of the acquired reflection and transmission channel images. Step 8: Calculate the correlation function of different combinations of the two channel image sequences after localization by the subpixel localization algorithm, and form a system of equations to solve the corresponding monochromatic correlation function to separate the signals of different wavelengths; Step 9: Use a multi-channel iterative method to reduce crosstalk in the separated monochromatic wavelength signals; Step 10: Use the images of different wavelengths after reducing color bleeding to perform super-resolution algorithm processing to obtain high-resolution images of different cell structures.

2. The multicolor fluorescence fluctuation imaging system based on a slowly varying transmittance dichroic mirror according to claim 1, characterized in that, The first dichroic mirror is selected to reflect the excitation light from the first converging lens and transmit the fluorescence emitted by the six marked quantum dots at wavelengths that are also ...

3. The multicolor fluorescence fluctuation imaging system based on a slowly varying transmittance dichroic mirror according to claim 1, characterized in that, Gradually varying transmittance is expressed using an expression for the Sigmoid function: （1） In the formula: λ is the wavelength; a and b are the slope parameters of the curve.

4. The multicolor fluorescence fluctuation imaging system based on a slowly varying transmittance dichroic mirror according to claim 1, characterized in that, Multiple sets of n-order correlation functions were obtained by combining different methods on the subpixel aligned dual-channel fluorescence signal image; the reflected and transmitted fluorescence signals generated by spectral dispersion through a second dichroic mirror based on the slowly varying transmittance of the Sigmoid function are represented as follows: （2） （3） In the formula: r represents the pixel coordinates; t represents the signal acquisition time; Indicates the reflected channel signal and Indicates the transmission channel signal; This represents the reflectivity of the dichroic mirror for the i-th wavelength signal; The transmittance and reflectance of the dichroic mirror for the i-th wavelength signal are represented by . With transmittance The theoretical value of the sum is 1; Calculate the nth-order correlation function for the fluorescence fluctuation signals of the two channels. ,get: （4） In the formula: Represents pixel coordinates; N represents the signal acquisition time; This indicates that the dichroic mirror is related to the first... The reflectivity of a signal of a specific wavelength; This indicates that the dichroic mirror is related to the first... Transmittance of a single wavelength signal; Indicates the first The intensity signal distribution of a type of fluorescent molecule; Indicates the first Monochromatic correlation function of wavelength fluorescence fluctuation signal; calculation of the combination of reflection channel signal and transmission channel signal. The order correlation function can be obtained One equation; when the number of wavelength channels equal When the number of unknowns in the system of equations equals the number of equations, the monochromatic correlation function can be directly obtained by solving the equations. That is, extracting the signal for each wavelength; The separated images at different wavelengths are processed to reduce crosstalk, resulting in individual images of each wavelength of the multicolor biological cell sample. The iterative formula for reducing crosstalk is shown below: （5） In the formula: λ is the color mixing factor; λ is the scaling factor, and its value range is 0 < λ < 1.

Images