CN-116774418-B - Multicolor fluorescence fluctuation imaging system and method based on slow-varying transmittance dichroic mirror

CN116774418BCN 116774418 BCN116774418 BCN 116774418BCN-116774418-B

Abstract

The invention relates to a multicolor fluorescence fluctuation imaging system and method based on a graded transmittance dichroic mirror, wherein the system comprises the steps of marking different subcellular organelle structures by adopting various wavelength quantum dot fluorescence, exciting by using single wavelength excitation light to obtain multicolor fluorescence signals, carrying out light splitting on the multicolor fluorescence signals based on the graded transmittance dichroic mirror, simultaneously collecting the multicolor fluorescence signals of a reflection channel and a transmission channel by using a single camera, separating the fluorescence signals of different wavelengths by adopting a method based on high-order correlation function calculation, reducing cross color among different channels by adopting a method of mutually iterating multi-wavelength channel signals, and carrying out super-resolution algorithm processing on images of different wavelengths after cross color reduction by adopting a super-resolution imaging method based on fluorescence fluctuation to obtain high-resolution images of different subcellular organelle fine structures. Finally, through single-phase machine acquisition of single-wavelength excitation and double-channel signals, separation of multicolor fluorescence signals, synchronous multicolor super-resolution imaging of different subcellular organelle structures is realized.

Inventors

ZENG ZHIPING
LI WENBO
QIU JIN
XU BIQING
XU CANHUA
HUANG YANTANG

Assignees

福州大学

Dates

Publication Date: 20260512
Application Date: 20230621

Claims (4)

1. The multicolor fluorescence fluctuation imaging system based on the slow-changing transmittance dichroic mirror is characterized by comprising an excitation light source for providing a beam of excitation light; a first converging lens for condensing the excitation light from the excitation light source, and the excitation light from the excitation light source penetrates the first converging lens; a first dichroic mirror reflecting the excitation light from the first focusing lens; An objective lens for transmitting the excitation light from the first dichroic mirror; A stage on which a biological cell sample is disposed, and on which the excitation light from the objective lens is irradiated, the biological cell sample emitting fluorescence under excitation of the excitation light from the objective lens, the fluorescence being emitted to the first dichroic mirror through the objective lens, the first dichroic mirror transmitting the fluorescence from the objective lens; A second condensing lens condensing the fluorescence from the first dichroic mirror, and the fluorescence from the first dichroic mirror penetrates the second condensing lens; A third condensing lens condensing the fluorescence from the second condensing lens, and the fluorescence from the second condensing lens penetrates the third condensing lens; A second dichroic mirror for splitting the fluorescence from the third converging lens, a part of the fluorescence from the third converging lens being reflected by the second dichroic mirror, and another part of the fluorescence from the third converging lens being transmitted by the second dichroic mirror; A first mirror that reflects the fluorescence reflected by the second dichroic mirror; a second mirror reflecting the fluorescent light reflected by the first mirror; a third mirror for reflecting the fluorescent light reflected by the second mirror; a fourth reflecting mirror that reflects the fluorescence transmitted by the second dichroic mirror; a fifth reflecting mirror for reflecting the fluorescence reflected by the fourth reflecting mirror; A sixth reflecting mirror for reflecting the fluorescence reflected by the fifth reflecting mirror; a seventh reflecting mirror for reflecting the fluorescence reflected by the sixth reflecting mirror; a CMOS camera for imaging the fluorescence reflected from the third mirror and for imaging the fluorescence reflected from the seventh mirror; The imaging method comprises the following steps: Setting a synchronous multicolor fluorescence fluctuation super-resolution imaging system based on a slow-changing transmittance dichroic mirror, wherein the synchronous multicolor fluorescence fluctuation super-resolution imaging system based on the slow-changing transmittance dichroic mirror comprises an excitation light source, a first converging lens, a first dichroic mirror, an objective lens, an objective table, a second converging lens, a third converging lens, a second dichroic mirror based on the slow-changing 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, respectively marking different cell structures in a biological cell sample by using six quantum dots with different fluorescence emission wavelengths, and carrying out short-wave excitation luminescence on the six quantum dots by using single-wavelength excitation light; Step 3, placing and fixing the marked biological cell sample on an objective table; step 4, adjusting the distance between the objective lens and the biological cell sample to enable the biological cell sample to be positioned on the focal point of the objective lens, and enabling the imaging system to meet the object-image conjugation relation of the optical microscope system; The exciting light emitted by the exciting light source is emitted to the first dichroic mirror through the first converging lens, the light reflected by the first dichroic mirror irradiates the biological cell sample through the object lens, the biological cell sample is stimulated to generate fluorescence, the fluorescence is emitted to the first dichroic mirror through the object lens, the light transmitted by the first dichroic mirror is emitted to the second converging lens to be converged, the light transmitted by the second converging lens is emitted to the third converging lens to be converged, the light transmitted by the third converging lens is emitted to the second dichroic mirror, the light reflected by the second dichroic mirror is reflected to the second reflecting mirror through the first reflecting mirror, the light reflected by the second reflecting mirror is reflected to the CMOS camera to perform fluorescence imaging, the light transmitted by 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, and the light reflected by the seventh reflecting mirror is reflected to the CMOS camera to perform fluorescence imaging; step 6, using and adjusting the output power of single-wavelength excitation light, exciting a plurality of wavelength quantum dots to emit light at the same time, observing and collecting fluorescent images by using a single camera, and stabilizing the output power of the excitation light when an obvious fluorescent intensity flickering phenomenon is observed; Step 7, synchronously collecting signals of a reflection channel and a transmission channel which are split by a Sigmoid curve gradual transmittance dichroic mirror by using a single camera, and positioning the same region of the collected image time sequence of the reflection channel and the transmission channel by using a sub-pixel positioning algorithm; Step 8, calculating correlation functions of different combinations by using two channel image sequences positioned by a sub-pixel positioning algorithm, forming an equation set to solve corresponding monochromatic correlation functions, and separating out signals with different wavelengths; Step 9, performing cross color reduction treatment on the separated monochromatic wavelength signals by using a multichannel mutual iteration method; And 10, performing super-resolution algorithm processing by using the pictures with different wavelengths after cross color reduction, and obtaining high-resolution images with different cell structures.
2. The system according to claim 1, wherein the first dichroic mirror is selected from wavelengths capable of reflecting excitation light from the first focusing lens and transmitting fluorescence light emitted from the six kinds of marked quantum dots, the transmittance of the second dichroic mirror is a gradual transmittance based on a Sigmoid curve, the polychromatic fluorescence signal is split and has different reflection and transmission response coefficients for fluorescence signals of different wavelengths, and the first, second, third, fourth, fifth, sixth and seventh reflecting mirrors reflect reflected light and transmitted light generated by the second dichroic mirror split onto the same CMOS camera.
3. The system for imaging polychromatic fluorescence fluctuation based on a slow-varying transmittance dichroic mirror according to claim 1, wherein the slow-varying transmittance is represented by an expression of Sigmoid function: (1) wherein: Wavelength, and a and b are curve slope parameters.
4. The system of claim 1, wherein the multiple sets of n-order correlation functions are obtained by combining the two-channel fluorescence signal pictures aligned by sub-pixels, and the reflected and transmitted fluorescence signals generated by the second dichroic mirror based on the Sigmoid function is expressed as: (2) (3) wherein r represents pixel point coordinates, t represents signal acquisition time; representing reflected channel signals Representing a transmission channel signal; indicating the reflectivity of the dichroic mirror to the i-th wavelength signal; Indicating the transmittance and reflectance of the dichroic mirror for the ith wavelength signal Transmittance and transmittance of And a theoretical value of 1; Calculating an n-order correlation function for two-channel fluorescence fluctuation signals The method comprises the following steps of: (4) wherein: Representing pixel coordinates; Representing the time of signal acquisition N Indicating the dichroic mirror pair of Reflectivity of the individual wavelength signals; indicating the dichroic mirror pair of Transmittance of the individual wavelength signals; Represent the first Intensity signal distribution of the seed fluorescent molecules; Represent the first Single-color correlation function of wavelength fluorescence fluctuation signal, calculation of combination of reflection channel signal and transmission channel signal The order correlation function can be obtained Equation when the number of wavelength channels Equal to When the number of unknowns in the equation set is equal to the number of equations, the monochromatic related function is directly solved by solving the equations I.e. extracting the signal of each wavelength; And performing cross color reduction treatment on the separated pictures with different wavelengths to obtain independent images of each wavelength of the multicolor biological cell sample, wherein an iterative formula for reducing cross color is as follows: (5) wherein: And lambda is a scaling factor, and the value range of lambda is 0< lambda <1.

Description

Multicolor fluorescence fluctuation imaging system and method based on slow-varying transmittance dichroic mirror Technical Field The invention relates to the technical field of an optical imaging system and a synchronous multicolor fluorescence separation method, in particular to a multicolor fluorescence fluctuation imaging system and a method based on a slow-varying transmittance dichroic mirror. Background The fluorescence fluctuation super-resolution technology utilizes the fluctuation characteristic of fluorescence signals to break through the diffraction limit of the traditional optical microscopic imaging. The existing super-resolution microscopic imaging technology based on fluorescence fluctuation, such as super-resolution optical scintillation microscopic imaging technology (SOFI), super-resolution radial fluctuation algorithm (SRRF), multi-signal classification algorithm (MUSICAL) based on fluorescence fluctuation, mean shift super-resolution algorithm (MSSR) and the like, can remarkably improve the imaging resolution. The existing multicolor fluorescence microscopic imaging mainly carries out multicolor image acquisition in a mode of spatial wavelength division or temporal wavelength division. The method comprises the steps of receiving a multi-color sample, and acquiring fluorescence of different wavelengths in different time periods by time according to the multi-color sample, wherein the time is different between different wavelengths, and the time resolution of imaging is obviously reduced. The multi-wavelength excitation and high-order cumulant separation multi-color spectrum technology requires multiple excitation light paths, so that the complexity of a system is remarkably increased, the stability of the system is reduced, and the high-order cumulant function analysis method can cause multiple artifact signals to influence the quality of image reconstruction. The existing polychromatic super-resolution imaging technology can realize signal separation of multi-wavelength signals usually through a method of spatial wavelength division acquisition or time wavelength division in the process of processing an image sequence. The spatial division wavelength acquisition of the signal greatly increases the complexity and cost of the imaging system, and the temporal division wavelength reduces the imaging rate. Therefore, the problem of mutual restriction among the number of excitation light paths, the number of occupied space wavelength-division channels and the elimination of channel delay in further development of multicolor fluorescence microscopy technology limits the application of fluorescence fluctuation-based super-resolution microscopy imaging in life science research such as subcellular organelles for synchronously observing multicolor marks. Disclosure of Invention In view of the above, the present invention aims to provide a system and a method for imaging polychromatic fluorescence fluctuation based on a dichroic mirror with a slow-varying transmittance, which can collect and separate polychromatic fluorescence samples under the condition of using only single-wavelength excitation light, two fluorescence signal collection channels and single-camera image collection, and effectively solve the problem of mutual restriction among the number of excitation light paths, the number of occupied space wavelength division channels and the elimination of channel delay. The multicolor fluorescence fluctuation imaging system based on the slow-changing transmittance dichroic mirror comprises an excitation light source for providing a beam of excitation light; a first converging lens for condensing the excitation light from the excitation light source, and the excitation light from the excitation light source penetrates the first converging lens; a first dichroic mirror reflecting the excitation light from the first focusing lens; An objective lens for transmitting the excitation light from the first dichroic mirror; A stage on which a biological cell sample is disposed, and on which the excitation light from the objective lens is irradiated, the biological sample emitting fluorescence under excitation of the excitation light from the objective lens, the fluorescence being emitted to the first dichroic mirror through the objective lens, the first dichroic mirror transmitting the fluorescence from the objective lens; A second condensing lens condensing the fluorescence from the first dichroic mirror, and the fluorescence from the second dichroic mirror penetrates the second condensing lens; A third condensing lens condensing the fluorescence from the second condensing lens, and the fluorescence from the second condensing lens penetrates the third condensing lens; A second dichroic mirror for splitting the fluorescence from the third converging lens, a part of the fluorescence from the third converging lens being reflected by the second dichroic mirror, and another part of the fluorescence from the