CN116337829A - High-channel fluorescence radiation differential microscopic imaging method and device - Google Patents

Abstract

The invention discloses a high-channel fluorescent radiation differential microscopic imaging method and device, which comprises a laser, a beam splitter, an optical fiber mode selection module, an optical field adjustment module, a microscopic imaging module, a detection module and a control unit, wherein N hollow excitation light spots and N solid excitation light spots which are overlapped in space positions are time-sharing gated by the optical fiber mode selection module, N independent FED microscopic imaging channels are formed by the N excitation light spots, the optical field adjustment module, the microscopic imaging module and N detection optical fibers contained in the detection module in a one-to-one correspondence manner, so that structural information of a sample to be detected can be obtained in parallel, and the imaging speed of FED is improved.

Description

High-channel fluorescence radiation differential microscopic imaging method and device

Technical Field

The invention belongs to the super-resolution imaging technology, and particularly relates to a high-channel fluorescence radiation differential (Fluorescence Emission Difference, FED) microscopic imaging method and device.

Background

The optical microscope has very important roles in the fields of life science, material science and the like, and can intuitively observe the microstructure of a sample. However, the spatial resolution of an optical microscope is typically greater than λ/2, limited by the optical diffraction limit. For this reason, super-resolution technologies such as structured light illumination microscopy, random optical reconstruction microscopy, stimulated radiation loss microscopy and the like have emerged continuously for decades. The fluorescence radiation differential (FED) microscopy technology uses digital processing to obtain two images of solid spot excitation and hollow spot excitation, so that super-resolution microscopic images can be obtained. Moreover, the technology has very good universality, and has lower requirements on fluorescent dye, fluorescent mark, laser power and the like compared with other super-resolution technologies.

In the early stage, the FED microscopic imaging technology firstly utilizes Gaussian solid excitation light spots to obtain a fluorescence confocal microscopic image of one sample, then utilizes donut type hollow light spots to obtain a fluorescence confocal microscopic image of the other sample, and finally reconstructs a super-resolution image through a digital processing technology. Because confocal microscopic imaging of a sample in a two-time point scanning mode is carried out, the imaging speed is slow, and therefore, the FED microscopic imaging quality is easily affected by factors such as vibration, temperature and the like of an imaging environment.

In order to improve the imaging speed, the array detector is used for receiving fluorescent signals of a sample, signals received by the central area of the array detector correspond to virtual solid spot excitation signals through signal processing, signals received by the edge area correspond to fluorescent signals excited by virtual hollow spots, and FED super-resolution images can be obtained quickly.

By staggering the incidence angles of the solid spot excitation light beam and the hollow spot excitation light beam, a pair of solid spot excitation light beam and hollow spot excitation light beam with certain deviation in spatial position can be formed in the sample at the same time, and two independent detectors are utilized to respectively receive fluorescent signals excited by the solid spot excitation light beam and the hollow spot excitation light beam, so that the speed of single-channel FED microscopic imaging can be improved by 2 times.

The polarization state of the incident excitation light is selectively modulated by a Spatial Light Modulator (SLM), s component and p component of the same excitation light are respectively incident into different spatial regions of the SLM to carry out different modulation, and a pair of solid excitation spots and hollow excitation spots with certain deviation of spatial positions can be obtained in a sample at the same time, so that single-channel rapid FED microscopic imaging with more stable optical path system and more convenient adjustment is realized.

However, all the above methods belong to single-channel FED microscopy imaging, and the improvement of imaging speed is still limited.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a high-channel fluorescence radiation differential microscopic imaging method and device, which form multichannel parallel FED microscopic imaging and improve the FED microscopic imaging speed.

To achieve the above object, an embodiment provides a high-channel fluorescence radiation differential microscopic imaging device, including:

a laser (1) for providing at least one excitation beam (8) of a central wavelength;

a beam splitter (2) for splitting the excitation beam (8) into excitation sub-beams (9) and (10) and directing the excitation sub-beams to two excitation light transmission channels, solid spot excitation and hollow spot excitation of the fiber mode selection module (3);

the optical fiber mode selection module (3) is used for time-sharing gating the two excitation light transmission channels, and outputting multiple independent solid spot excitation light beams and hollow spot excitation light beams by the optical fiber array;

the optical field adjusting module (4) is used for carrying out optical field adjustment on the multipath light beams output by the optical fiber mode selecting module (3) so as to form a linear excitation light spot array; and is also used for imaging the multipath fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6);

the microscopic imaging module (5) is used for imaging the linear excitation light spot array to a sample to be detected and collecting fluorescent signals generated by excitation of the sample;

the detection module (6) is used for receiving multiple paths of fluorescent signals in parallel by adopting the detector array and converting the multiple paths of fluorescent signals into electric signals;

the control unit (7) is used for controlling the optical fiber mode selection module (3) and the microscopic imaging module (5) to work, and is also used for collecting the electric signals of the detection module (6) and performing super-resolution imaging;

n paths of hollow excitation light beams or N paths of solid excitation light beams are output in a time sharing mode through the optical fiber mode selection module (3), N paths of excitation light beams, the optical field adjustment module (4), the microscopic imaging module (5) and N detection optical fibers contained in the detection module (6) are in one-to-one correspondence to form N independent FED microscopic imaging channels, and therefore structural information of a sample to be detected is obtained in parallel.

In an alternative embodiment, the beam splitter (2) selects energy beam splitting or polarization beam splitting according to the polarization characteristics of the excitation light beam, when the energy beam splitting is selected, the beam splitter (2) adopts an energy beam splitter including a beam splitting prism or a beam splitter sheet, when the polarization beam splitting is selected, the beam splitter (2) adopts a polarization beam splitter including a polarization beam splitting prism, and when the laser (1) outputs with a pigtail, the beam splitter (2) adopts a fiber optic beam splitter of 1*2.

In an alternative embodiment, the optical fiber mode selection module (3) includes optical fiber mode selection units (11) and (12) corresponding to the two excitation light transmission channels of solid spot excitation and hollow spot excitation respectively, the optical fiber mode selection units (11) and (12) each include 1 acousto-optic modulator (AOM) and 1 optical fiber mode selector, the two AOMs are used as optical switches for respectively controlling gating time sequences of the solid spot excitation and the hollow spot excitation so as to realize high-speed switching of two excitation light spot modes, and the two optical fiber mode selectors are used for respectively selecting N solid spot output optical fibers and N hollow spot output optical fibers and respectively outputting N independent solid spot excitation light beams and N independent hollow spot excitation light beams in a time sharing way through AOM control;

the optical fiber mode selector unit (11) comprises an optical fiber mode selector which selects 1*N single-mode optical fiber beam splitters, 1*2 single-mode optical fiber coupler combinations of N output ends, 1*4 single-mode optical fiber coupler combinations of N output ends or 1*2 and 1*4 single-mode optical fiber coupler combinations of N output ends;

the optical fiber mode selection unit (12) comprises an optical fiber mode selector for selecting 1*N single-mode optical fiber beam splitters, N optical fiber mode selection couplers, N1*2 single-mode optical fiber coupler combinations of output ends, N optical fiber mode selection couplers, N1*4 single-mode optical fiber coupler combinations of output ends, N optical fiber mode selection couplers or N1*2 single-mode optical fiber couplers of output ends, and 1*4 single-mode optical fiber couplers, and N optical fiber mode selection couplers, wherein the optical fiber mode selection couplers are composed of single-mode optical fibers and few-mode optical fibers.

In an alternative embodiment, at the end face (21) of the optical fiber array included in the optical fiber mode selection module (3), an optical fiber fixture is used to fix the end faces of the N solid spot output optical fibers and the N hollow spot output optical fibers of the optical fiber mode selection module (3), so that each solid spot output optical fiber and each hollow spot output optical fiber form a group, and the excitation light point source can be controlled in a time-sharing manner corresponding to one FED microscopic imaging channel.

In an alternative embodiment, the light field adjustment module (4) comprises a beam collimation unit (22), a converging lens (23), a lens (25), a dichroic mirror (26), a lens (27), a converging lens (30);

the beam collimation unit (22) controls N solid spot excitation beams and N hollow spot excitation beams which are output by the optical fiber array end face (21) of the optical fiber mode selection module (3) to be collimated and output in parallel;

the converging lens (23) controls N solid spot excitation light beams or N hollow spot excitation light beams which are time-division gated to form a linear excitation light spot array (24) with overlapped space positions, wherein the linear excitation light spot array (24) consists of N solid excitation light spots or N hollow excitation light spots which are gated;

the lens (25) and the lens (27) control the linear excitation light spot array (24) to form a linear excitation light spot array (28), and the linear excitation light spot array (28) forms an image again through the microscopic imaging module (5) to realize linear excitation illumination;

the dichroic mirror (26) is arranged between the lens (25) and the lens (27) and is used for separating the excitation light beam and the fluorescence signal;

the lens (27), the dichroic mirror (26) and the converging lens (30) are used for sequentially carrying out shunt transmission on multiple paths of fluorescent signals obtained by the microscopic imaging module (5), the method comprises the steps of collimating N paths of independent fluorescent signals output by the microscopic imaging module (5) through the lens (27), separating excitation light beams and fluorescent signal light beams through the dichroic mirror (26), converging N paths of fluorescent signal light beams through the converging lens (30), and enabling the N paths of fluorescent signal light beams to enter the detection module (6).

In an alternative embodiment, the microscopic imaging module (5) comprises at least a scanning unit and a microscopic objective, wherein the microscopic objective is used for imaging the linear excitation light spot array (28) into the sample to be tested, forming the linear excitation light spot array (29), and receiving fluorescent signals generated by the excitation light field; the scanning unit is connected with the control unit (7) through a data line to realize external communication control and is used for guiding the linear excitation light spot array (29) to perform two-dimensional or three-dimensional scanning movement on the sample according to a scanning command of the control unit (7).

In an alternative embodiment, the beam collimation unit (22) selects a microlens array; the converging lens (23) selects a cylindrical lens.

In an alternative embodiment, the detection module (6) comprises an optical fiber array end face (31), a detection optical fiber array and a detector array, and multiple fluorescent signals received by the optical fiber array end face (31) are respectively led into the detector array through the detection optical fiber array to be converted into multiple electric signals.

In an alternative embodiment, the control unit (7) controls the optical fiber mode selection module (3) to work, and sends instructions to the optical fiber mode selection module (3) to gate the excitation light transmission channels of the solid spot excitation and the hollow spot excitation, and controls the imaging sequence corresponding to the solid spot excitation and the hollow spot excitation;

when the micro imaging module (5) is controlled by the control unit (7) to work, a scanning instruction is sent to a scanning unit of the micro imaging module (5), and the linear excitation light spot array (29) is controlled to perform two-dimensional/three-dimensional scanning on a sample;

when the control unit (7) collects the electric signals of the detection module (6) and performs super-resolution imaging, the super-resolution microscopic image of the sample is reconstructed and displayed by using the FED technology after the solid spot excitation sample image and the hollow spot excitation sample image which are obtained based on the electric signals are obtained according to the scanning time sequence.

To achieve the above object, an embodiment further provides a high-channel fluorescence radiation differential microscopic imaging method, which adopts the device, and includes the following steps:

-providing at least one excitation beam (8) of a central wavelength by means of a laser (1);

dividing an excitation beam (8) into excitation sub-beams (9) and (10) by a beam splitter (2) and guiding the excitation sub-beams into two excitation light transmission channels, namely solid spot excitation and hollow spot excitation, of the optical fiber mode selection module (3);

the optical fiber mode selection module (3) is used for time-sharing gating the two excitation light transmission channels, and a fiber array is used for outputting multiple independent solid spot excitation light beams and hollow spot excitation light beams;

the optical field adjustment module (4) is used for carrying out optical field adjustment on the multipath light beams output by the optical fiber mode selection module (3) so as to form a linear excitation light spot array; and is also used for imaging the multipath fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6);

imaging the linear excitation light spot array to a sample to be detected through a microscopic imaging module (5), and collecting fluorescent signals generated by excitation of the sample;

the detector array contained by the detection module (6) receives multiple paths of fluorescent signals in parallel and converts the fluorescent signals into electric signals;

the optical fiber mode selection module (3) and the microscopic imaging module (5) are controlled to work through the control unit (7), and the electrical signals of the detection module (6) are collected and super-resolution imaging is carried out.

Compared with the prior art, the invention has the beneficial effects that at least the following steps are included:

1) The optical fiber mode selection module is excited by multiple paths of solid light spots and hollow light spots in a time-sharing manner, and the array detector combined with the detection module receives and forms multiple paths of FED microscopic imaging channels in parallel, so that the imaging speed of the FED can be improved.

2) The excitation channel is selected by AOM at high speed in a time sharing way, and the excitation light mode (solid light spot and hollow light spot) is selected by the optical fiber, so that the light path structure of the device is more stable and compact, and the influence of the external environment on FED microscopic imaging is reduced.

3) The optical fiber clamp and the optical field adjusting module are utilized to enable the spatial positions of each group of hollow and solid excitation light spots to be overlapped, so that the field of view of FED microscopic imaging can be improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a high-channel fluorescence radiation differential microscopic imaging device provided in an embodiment;

FIG. 2 is a schematic diagram of the structure of an end face of an excitation fiber array provided in an embodiment;

FIG. 3 is a schematic diagram of the structure of an end face of a detection fiber array according to an embodiment;

FIG. 4 is a flow chart of a high-channel fluorescence radiation differential microscopy imaging method provided by the embodiment.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the detailed description is presented by way of example only and is not intended to limit the scope of the invention.

As shown in fig. 1, the high-channel FED microscopic imaging device provided in the embodiment includes a laser 1, a beam splitter 2, an optical fiber mode selection module 3, a light field adjustment module 4, a microscopic imaging module 5, a detection module 6, and a control unit 7. The imaging basic principle of the device is FED microscopic imaging technology, two confocal fluorescence microscopic images obtained through solid light spot excitation and hollow light spot excitation are utilized to obtain super-resolution fluorescence information by utilizing a difference technology. The device is structurally arranged, wherein N paths of hollow excitation light beams or N paths of solid excitation light beams are output in a time-sharing mode through the light mode selection module 3, N paths of excitation light beams, the light field adjustment module 4, the microscopic imaging module 5 and N detection optical fibers contained in the detection module 6 are in one-to-one correspondence to form N independent FED microscopic imaging channels, so that structural information of a sample to be detected can be obtained in parallel, and the imaging speed of an FED can be improved. The key is that the two AOMs included in the optical fiber mode selection module 3 gate two excitation light transmission channels in a high-speed time-sharing manner, so that N paths of hollow excitation light beams and N paths of solid excitation light beams can be stably output, the optical field adjustment module 4 enables the space positions of each group of gated hollow excitation light spots and solid excitation light spots to be transmitted in a highly overlapped manner, and the end faces of the excitation fibers included in the optical fiber mode selection module 3 and the end faces of the detection fibers included in the detection module 6 are aligned to form N paths of excitation light output ports and N paths of signal light detection ports of the multi-path FED imaging channel. The following details for each section:

in an embodiment, the laser 1 is configured to provide at least one excitation beam 8 with a central wavelength, i.e. to provide a single or multi-color excitation beam, which can make the invention better suited for different optical characteristics of the sample to be examined in imaging principle, and to implement high-throughput FED microscopy imaging of one or more fluorescent dye markers, so that the dynamic information of the sample to be examined can be studied or observed more comprehensively. Preferably, the laser 1 may be selected from a single wavelength laser, a wavelength tuned laser or a white light laser. When the output beam of the laser 1 is output to free space, a beam expander composed of a telescopic system is required to collimate the output beam into a parallel beam.

In an embodiment, the beam splitter 2 is configured to split the excitation beam 8 into excitation sub-beams 9 and 10 and direct the excitation sub-beams into two excitation light transmission channels, solid spot excitation and hollow spot excitation, of the fiber mode selection module 3. For the collimated parallel excitation beam output by the laser 1, the beam splitter 2 may select energy beam splitting or polarization beam splitting according to the polarization characteristics of the excitation beam, when the energy beam splitting is selected, the beam splitter 2 adopts an energy beam splitter including a beam splitting prism or a beam splitter, when the polarization beam splitting is selected, the beam splitter 2 adopts a polarization beam splitter including a polarization beam splitting prism, and for the laser 1, the excitation beam is output by adopting a pigtail, and the beam splitter 2 adopts a fiber beam splitter of 1*2. In particular, when an energy beam splitter is employed, the energy beam splitter splits the excitation beam 8 into excitation sub-beams 9 and 10 at an energy ratio of 1:1.

In the embodiment, the optical fiber mode selection module 3 is used for time-sharing gating two excitation light transmission channels, and multiple independent solid-spot excitation light beams and hollow-spot excitation light beams are output by the optical fiber array. The optical fiber mode selection module 3 includes optical fiber mode selection units 11 and 12 corresponding to the two excitation light transmission channels of solid spot excitation and hollow spot excitation, respectively. The optical fiber mode selection units 11 and 12 respectively comprise 1 acousto-optic modulator (AOM) and 1 optical fiber mode selector, wherein the two AOMs are used as optical switches for respectively controlling the working time of the excitation light beam entering the two excitation transmission channels, namely respectively controlling the gating time sequence of solid spot excitation and hollow spot excitation so as to realize high-speed switching of two excitation light spot modes, and the two optical fiber mode selectors are used for respectively selecting N solid spot output optical fibers and N hollow spot output optical fibers and respectively outputting N independent solid spot excitation light beams and N independent hollow spot excitation light beams in a time-sharing way through the AOM control.

The optical fiber mode selector included in the optical fiber mode selecting unit 11 selects 1*N single-mode optical fiber beam splitters, 1*2 single-mode optical fiber coupler combinations of N output ends, 1*4 single-mode optical fiber coupler combinations of N output ends, or 1*2 and 1*4 single-mode optical fiber coupler combinations of N output ends, so as to output N solid light spot excitation light beams.

The optical fiber mode selecting unit 12 includes an optical fiber mode selector for selecting 1*N single-mode optical fiber beam splitters and N optical fiber mode selecting couplers, N1*2 single-mode optical fiber coupler combinations and N optical fiber mode selecting couplers of N output ends, N1*4 single-mode optical fiber coupler combinations and N optical fiber mode selecting couplers of N output ends, or N1*2 single-mode optical fiber coupler combinations and 1*4 single-mode optical fiber coupler combinations and N optical fiber mode selecting couplers of N output ends, so as to output N hollow light spot excitation light beams. The single-mode fiber mode selection coupler is composed of a single-mode fiber and a few-mode fiber, the single-mode fiber directly controls and outputs a single-channel solid spot excitation beam, and the single-mode fiber mode selection coupler controls and can output the single-channel hollow spot excitation beam.

The optical fiber coupler is used for dividing the laser beam into multiple excitation beams with equal intensity according to the energy ratio, the optical fiber beam splitter of 1*2 outputs two excitation light transmission channels, preferably an optical fiber input/output type AOM, and controls the gating time sequence of solid spot excitation and hollow spot excitation. For solid excitation modes, a 1*N fiber coupler preferably formed by single-mode fibers outputs N independent solid excitation beams; for the hollow excitation mode, an optical fiber mode selection coupler composed of a single-mode optical fiber and a few-mode optical fiber is preferable, and an independent hollow excitation beam is output from N few-mode optical fibers.

To simplify the structure of the system optical path and improve the stability of the system, the AOM preferably employs optical fiber input and output. As shown in fig. 2, in the system, the end faces of the N solid spot output fibers and the N hollow spot output fibers of the optical fiber mode selection module 3 are fixed by using an optical fiber fixture, so that each solid spot output fiber and each hollow spot output fiber form a group, and the excitation light point sources can be controlled in a time-sharing manner, corresponding to one FED microscopic imaging channel. In embodiments, a low coefficient of thermal expansion fiber optic clamp is preferred.

Specifically, for example, as shown in fig. 1 and 2, the fiber mode selection unit 11 outputs 4 independent solid spot excitation light beams 13, 14, 15, 16, and the fiber mode selection unit 12 outputs 4 independent donut type hollow spot excitation light beams 17, 18, 19, 20. The optical fiber bundles for transmitting 4 solid light spot excitation light beams 13, 14, 15, 16 and hollow light spot excitation light beams 17, 18, 19, 20 are fixed on the end face 21 of the optical fiber array by using an optical fiber fixture, so that every two optical fibers for outputting the hollow light spot and the solid light spot form a group, for example, 13 and 17 form a group, and a group corresponds to an excitation light source of an FED microscopic imaging channel.

In the embodiment, the optical field adjusting module 4 is configured to collimate and adjust the optical field of the multiple light beams output by the optical fiber mode selecting module 3 to form N parallel excitation light spot arrays with linear distribution, and is further configured to image multiple fluorescent signals obtained from the microscopic imaging module 5 to the detecting module 6. As shown in fig. 1, the light field adjustment module 4 includes a light beam collimation unit 22, a converging lens 23, a lens 25, a dichroic mirror 26, a lens 27, and a converging lens 30.

The beam collimation unit 22 controls the N solid spot excitation beams and the N hollow spot excitation beams which are output by the optical fiber array end face 21 to be mutually parallel and collimated and output. The converging lens 23 controls the N solid spot excitation light beams or the N hollow spot excitation light beams which are in time-sharing gating to form a linear excitation light spot array 24 with overlapped space positions, wherein the linear excitation light spot array 24 consists of the N solid excitation light spots which are in gating or the N hollow excitation light spots which are in gating. Lenses 25 and 27 provide appropriate magnification to control the imaging of the array of linear excitation spots 24 into the array of linear excitation spots 28 so that an array of linear excitation spots 29 with little interference of adjacent excitation spots can be obtained by the microimaging module 5.

A dichroic mirror 26 is provided between the lens 25 and the lens 27 for separating the excitation light beam and the fluorescence signal. The lens 27, the dichroic mirror 26 and the converging lens 30 sequentially perform shunt transmission on the multipath fluorescent signals obtained by the microscopic imaging module 5, specifically, N paths of independent fluorescent signals output by the microscopic imaging module 5 are collimated by the lens 27, the excitation light beam and the fluorescent signal light beam are separated by the dichroic mirror 26, and N paths of fluorescent signal light beams are converged by the converging lens 30 and enter the optical fiber array end face 31 of the detection module 6.

The beam collimation unit 22 is preferably a micro lens array according to the numerical aperture angle, the mode field radius, the optical fiber distance and the working wavelength of the hollow excitation light spot and the solid excitation light spot, and can collimate each group of hollow spot excitation light beam and solid spot excitation light beam and enable the output light beams of two light spot modes to be parallel to each other; the converging lens 23 is preferably a cylindrical lens capable of converging each of the groups of collimated hollow-spot excitation light beams and solid-spot excitation light beams at the same spatial location of the focal plane to form a linear excitation light spot array 24. The lens 25 and the lens 27 form an imaging system according to the entrance pupil and the field size of the microscopic imaging system, and the linear excitation light spot array 24 is imaged into a linear excitation light spot array 28 with proper size and interval; the lens 27 and the converging lens 30 constitute an imaging system for imaging the fluorescent signals output from the microscopic imaging module 5 onto the fiber array end face 31 of the detection module 6.

In an embodiment, the microscopic imaging module 5 is configured to image the linear excitation light spot array 28 onto the sample to be tested, and form a linear excitation light spot array 29 with a light spot pitch not smaller than one airy spot size, where the linear excitation light spot array 29 generates a multi-point parallel excitation area to excite the sample to be tested, the sample to be tested generates a fluorescent signal under excitation, and the fluorescent signal is received and input to the light field adjustment module 4 in an imaging manner.

Wherein the microscopic imaging module 5 comprises at least a scanning unit and a microscope objective. The microscope objective is used for imaging the linear excitation light spot array 28 into a sample to be detected, forming a linear excitation light spot array 29 with small adjacent excitation light spot interference, and receiving fluorescent signals generated by an excitation light field. The scanning unit is connected with the control unit 7 through a data line, and is used for realizing external communication control and guiding the linear excitation light spot array 29 to perform two-dimensional or three-dimensional scanning movement on the sample according to a scanning command of the control unit 7.

In the imaging principle, the illumination area of the linear excitation light spot array and the input end face of the linear arranged detection optical fiber array are conjugated, so that each excitation light spot and a corresponding detection optical fiber form a confocal imaging mode, namely an FED microscopic imaging channel. By time-sharing gating N solid spot excitation or N hollow spot excitation and combining scanning of a linear excitation light spot array, a sample microscopic image corresponding to solid spot excitation and a sample image corresponding to hollow spot excitation can be quickly obtained, and a super-resolution image of a sample, namely multichannel FED microscopic imaging, can be quickly reconstructed.

In an embodiment, the detection module 6 is configured to receive multiple fluorescence signals in parallel using a detector array and convert the multiple fluorescence signals into an electrical signal. As shown in fig. 1, the detection module 6 includes an optical fiber array end face 31, an array detection optical fiber array composed of detection optical fibers 32, 33, 34, 35, and a detector array composed of detectors 36, 37, 38, 39, and the optical fiber array end face 31 receives multiple fluorescent signals and splits the multiple fluorescent signals into multiple independent detection channels, and the multiple fluorescent signals received are respectively led into the detector array through the detection optical fiber array to be converted into multiple electrical signals. As shown in fig. 3, the array of detection fibers is arranged in a linear fashion.

The optical fiber array end face 31 is also fixed by adopting an optical fiber clamp, the structure of the optical fiber clamp is designed according to the numerical aperture, the outer diameter and the mode spot radius of the excitation and receiving optical fibers and the imaging resolution and the imaging range determined by the optical field adjusting module 4 and the microscopic imaging module 5, so that the solid excitation light spots and the hollow excitation light spots in each FED microscopic imaging channel are overlapped in space, the distance between the excitation light spots corresponding to adjacent FED microscopic imaging channels in a sample is at least greater than one Airy spot diameter, and the fluorescent signals of each FED microscopic imaging channel are efficiently coupled into the detection optical fiber array through the microscopic imaging module 5 and the optical field adjusting module 4.

The detection fibers 32, 33, 34, 35 are preferably multimode fibers for improved signal-to-noise ratio. When a plurality of independent detectors are adopted to receive signals, each detection optical fiber is respectively and directly connected with an end face optical fiber interface of the corresponding detector. The detector 36, 37, 38, 39 is selected to have N individual detectors or an area array detector comprising N detection units. When an area array detector comprising N detection units is used, the detection module 6 needs to be configured with at least a telecentric optical imaging system according to the area of the detection units detected by the area array, the mode radius and the numerical aperture of the detection optical fibers, and each path of signal output by the detection optical fibers 32, 33, 34, 35 can be received by the area array detector 36, 37, 38, 39 with a proper signal-to-noise ratio through the control of the telecentric optical imaging system. N independent detectors preferably select PMT or APD with N receiver end faces capable of being directly connected with optical fibers, and the PMT or APD array detector has single photon detection capability, can image the fluorescence lifetime of a sample by combining photon counting, can further expand the functions of a system, and can image the fluorescence lifetime of the sample to be detected in an FED mode. An area array detector comprising N detection cells preferably selects an APD array or a multi-pixel photon counter (MPPC). The detection module 6 is connected with the control unit 7 through a data line to realize the functions of data acquisition and transmission.

In the embodiment, the control unit 7 includes a computer, a data acquisition card, a scanning control card, a scanner driving circuit and an AOM driving circuit, and is used for controlling the optical fiber mode selection module 3 and the microscopic imaging module 5 to work, and is also used for acquiring the electric signals of the detection module 6 and performing super-resolution imaging. Specifically, the control unit 7 controls the optical fiber mode selection module 3 to operate, and sends an excitation light spot mode switching instruction to the AOM of the optical fiber mode selection module 3 to gate the excitation light transmission channels of solid spot excitation and hollow spot excitation, and controls the imaging sequences corresponding to the solid spot excitation and the hollow spot excitation. When the control unit 7 controls the microscopic imaging module 5 to work, a scanning instruction is sent to a scanning unit of the microscopic imaging module 5, and the linear excitation light spot array 29 is controlled to perform two-dimensional/three-dimensional scanning on the sample. When the control unit 7 collects the electric signals of the detection module 6 and performs super-resolution imaging, the sample microscopic image corresponding to solid spot excitation and the sample microscopic image corresponding to hollow spot excitation obtained based on the electric signals are obtained according to a scanning time sequence, and then the super-resolution microscopic image of the sample is reconstructed and displayed by using an FED technology.

With the imaging device, a high-flux FED microscopic imaging method uses a laser 1 to provide at least one single-color irradiation beam, and leads the beam to two transmission channels of an optical fiber mode selection module 3 through a beam splitter 2; the two AOMs of the optical fiber mode selection module 3 are used for selecting solid spot excitation channels and hollow spot excitation channels in a time-sharing mode, and the combination of the optical fiber coupler and the optical fiber mode selection coupler is used for respectively outputting N solid excitation light beams and N hollow excitation light beams; 2N excitation fiber end faces of the fiber mode selection module 3 are fixed by using a fiber clamp, so that each solid excitation spot output end and each corresponding hollow excitation spot output end form a group; the light field adjusting module 4 collimates N solid excitation light beams or N hollow excitation light beams which are time-division gated, so that the space positions of each group of solid spots and hollow spots are overlapped to form a linear excitation light spot array; imaging the linear excitation light spot array into the sample by using a microscopic imaging module 5, and receiving fluorescent signals of an excited region in the sample; the light field adjusting module 4 collimates, splits and focuses each independent fluorescent signal output by the microscopic imaging module 5 to the input end face of the detection optical fiber array; n detection fibers are directly connected with the input end faces of the separated N detectors, or the output end faces of the detection fibers are imaged on the receiving face of the area array detector by a telecentric imaging system; the scanning unit of the microscopic imaging module 5 is used for enabling the linear excitation light spot array 29 to perform two-dimensional or three-dimensional scanning movement in the sample, and image information of the sample is obtained in parallel through multiple channels; and a control unit 7 is used for sending a time gating instruction to the AOM, sending a scanning instruction to the scanning unit, collecting an electric signal output by the detection module, and reconstructing and displaying a super-resolution image of the sample according to the FED technology.

Embodiments also provide a high-channel fluorescence radiation differential microscopy imaging method employing the apparatus shown in fig. 1, as shown in fig. 4, comprising the steps of:

providing at least one excitation beam 8 of central wavelength by means of a laser 1;

the excitation beam 8 is divided into excitation sub-beams 9 and 10 by the beam splitter 2 and is led to two excitation light transmission channels of solid spot excitation and hollow spot excitation of the optical fiber mode selection module 3;

the optical fiber mode selection module 3 is used for time-sharing gating the two excitation light transmission channels, and a fiber array is used for outputting multiple independent solid spot excitation light beams and hollow spot excitation light beams;

the optical field adjustment module 4 is used for carrying out optical field adjustment on the multipath light beams output by the optical fiber mode selection module 3 so as to form a linear excitation light spot array; and also for imaging the multiple fluorescence signals obtained from the microscopic imaging module 5 to the detection module 6;

imaging the linear excitation light spot array to a sample to be detected through a microscopic imaging module 5, and collecting fluorescent signals generated by excitation of the sample;

the detector array contained by the detection module 6 receives the multipath fluorescent signals in parallel and converts the multipath fluorescent signals into electric signals;

the optical fiber mode selection module 3 and the microscopic imaging module 5 are controlled to work through the control unit 7, and electric signals of the detection module 6 are collected and super-resolution imaging is carried out.

The device and the method provided by the embodiment utilize the time-sharing gating solid and hollow excitation light spot arrays to excite in parallel, and combine the synchronous parallel reception of the detector arrays to realize the FED microscopic imaging with high channel number. The AOM time-sharing gating solid and hollow excitation beam channels are used, solid and hollow excitation beams are generated by optical fiber mode selection, the optical fiber clamp is used for fixing the spatial positions of the excitation optical fiber array and the detection optical fiber array, and the detector array is used for receiving the fluorescent signal array generated by the excitation array, so that the time-sharing excitation of the solid and hollow excitation light spot array and the parallel detection of the array fluorescent signals are realized, and a multipath FED microscopic imaging channel is formed. The device uses fiber array transmission at excitation and detection end, on one hand makes the light path structure stable, on the other hand makes the solid and hollow excitation facula of every way FED microscopic imaging passageway common way to improve imaging accuracy and speed, help observing or measuring the dynamic process of cell.

The foregoing detailed description of the preferred embodiments and advantages of the invention will be appreciated that the foregoing description is merely illustrative of the presently preferred embodiments of the invention, and that no changes, additions, substitutions and equivalents of those embodiments are intended to be included within the scope of the invention.

Claims (10)

1. A high-pass fluorescence radiation differential microscopic imaging device, comprising:

a laser (1) for providing at least one excitation beam (8) of a central wavelength;

a beam splitter (2) for splitting the excitation beam (8) into excitation sub-beams (9) and (10) and directing the excitation sub-beams to two excitation light transmission channels, solid spot excitation and hollow spot excitation of the fiber mode selection module (3);

the optical fiber mode selection module (3) is used for time-sharing gating the two excitation light transmission channels, and outputting multiple independent solid spot excitation light beams and hollow spot excitation light beams by the optical fiber array;

the optical field adjusting module (4) is used for carrying out optical field adjustment on the multipath light beams output by the optical fiber mode selecting module (3) so as to form a linear excitation light spot array; and is also used for imaging the multipath fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6);

the microscopic imaging module (5) is used for imaging the linear excitation light spot array to a sample to be detected and collecting fluorescent signals generated by excitation of the sample;

the detection module (6) is used for receiving multiple paths of fluorescent signals in parallel by adopting the detector array and converting the multiple paths of fluorescent signals into electric signals;

the control unit (7) is used for controlling the optical fiber mode selection module (3) and the microscopic imaging module (5) to work, and is also used for collecting the electric signals of the detection module (6) and performing super-resolution imaging;

n paths of hollow excitation light beams or N paths of solid excitation light beams are output in a time sharing mode through the optical fiber mode selection module (3), N paths of excitation light beams, the optical field adjustment module (4), the microscopic imaging module (5) and N detection optical fibers contained in the detection module (6) are in one-to-one correspondence to form N independent fluorescent radiation differential microscopic imaging channels, so that structural information of a sample to be detected is obtained in parallel.

2. The high-channel fluorescence radiation differential microscopic imaging apparatus according to claim 1, wherein the beam splitter (2) selects energy beam splitting or polarization beam splitting according to polarization characteristics of the excitation beam, when energy beam splitting is selected, the beam splitter (2) adopts an energy beam splitter including a beam splitting prism or a beam splitter, when polarization beam splitting is selected, the beam splitter (2) adopts a polarization beam splitter including a polarization beam splitting prism, and when the laser (1) outputs with a pigtail, the beam splitter (2) adopts a fiber optic beam splitter of 1*2.

3. The high-channel fluorescence radiation differential microscopic imaging apparatus according to claim 1, wherein the optical fiber mode selection module (3) comprises optical fiber mode selection units (11) and (12) corresponding to the two excitation light transmission channels of solid spot excitation and hollow spot excitation respectively, the optical fiber mode selection units (11) and (12) each comprise 1 acousto-optic modulator (AOM) and 1 optical fiber mode selector, the two AOMs are both used as optical switches for respectively controlling gating time sequences of the solid spot excitation and the hollow spot excitation so as to realize high-speed switching of two excitation light spot modes, and the two optical fiber mode selectors are used for respectively selecting N solid spot output optical fibers and N hollow spot output optical fibers and respectively outputting N independent solid spot excitation light beams and N independent hollow spot excitation light beams in a time sharing way through AOM control;

the optical fiber mode selector unit (11) comprises an optical fiber mode selector which selects 1*N single-mode optical fiber beam splitters, 1*2 single-mode optical fiber coupler combinations of N output ends, 1*4 single-mode optical fiber coupler combinations of N output ends or 1*2 and 1*4 single-mode optical fiber coupler combinations of N output ends;

the optical fiber mode selection unit (12) comprises an optical fiber mode selector for selecting 1*N single-mode optical fiber beam splitters, N optical fiber mode selection couplers, N1*2 single-mode optical fiber coupler combinations of output ends, N optical fiber mode selection couplers, N1*4 single-mode optical fiber coupler combinations of output ends, N optical fiber mode selection couplers or N1*2 single-mode optical fiber couplers of output ends, and 1*4 single-mode optical fiber couplers, and N optical fiber mode selection couplers, wherein the optical fiber mode selection couplers are composed of single-mode optical fibers and few-mode optical fibers.

4. The high-channel fluorescence radiation differential microscopic imaging device according to claim 3, wherein at an optical fiber array end face (21) included in the optical fiber mode selection module (3), end faces of the N solid spot output optical fibers and the N hollow spot output optical fibers of the optical fiber mode selection module (3) are fixed by an optical fiber fixture, so that each solid spot output optical fiber and each hollow spot output optical fiber form a group, and the excitation light point source can be controlled in a time-sharing manner corresponding to one fluorescence radiation differential microscopic imaging channel.

5. The high-channel fluorescence radiation differential microscopic imaging device according to claim 1, wherein the light field adjustment module (4) comprises a beam collimation unit (22), a converging lens (23), a lens (25), a dichroic mirror (26), a lens (27), a converging lens (30);

the beam collimation unit (22) controls N solid spot excitation beams and N hollow spot excitation beams which are output by the optical fiber array end face (21) of the optical fiber mode selection module (3) to be collimated and output in parallel;

the converging lens (23) controls N solid spot excitation light beams or N hollow spot excitation light beams which are time-division gated to form a linear excitation light spot array (24) with overlapped space positions, wherein the linear excitation light spot array (24) consists of N solid excitation light spots or N hollow excitation light spots which are gated;

the lens (25) and the lens (27) control the linear excitation light spot array (24) to form a linear excitation light spot array (28), and the linear excitation light spot array (28) forms an image again through the microscopic imaging module (5) to realize linear excitation illumination;

the dichroic mirror (26) is arranged between the lens (25) and the lens (27) and is used for separating the excitation light beam and the fluorescence signal;

the lens (27), the dichroic mirror (26) and the converging lens (30) are used for sequentially carrying out shunt transmission on multiple paths of fluorescent signals obtained by the microscopic imaging module (5), the method comprises the steps of collimating N paths of independent fluorescent signals output by the microscopic imaging module (5) through the lens (27), separating excitation light beams and fluorescent signal light beams through the dichroic mirror (26), converging N paths of fluorescent signal light beams through the converging lens (30), and enabling the N paths of fluorescent signal light beams to enter the detection module (6).

6. The high-channel fluorescence radiation differential microscopic imaging device according to claim 1, wherein the microscopic imaging module (5) comprises at least a scanning unit and a microscopic objective, wherein the microscopic objective is used for imaging the linear excitation light spot array (28) into the sample to be measured, forming the linear excitation light spot array (29), and receiving the fluorescence signal generated by the excitation light field; the scanning unit is connected with the control unit (7) through a data line to realize external communication control and is used for guiding the linear excitation light spot array (29) to perform two-dimensional or three-dimensional scanning movement on the sample according to a scanning command of the control unit (7).

7. The high-channel fluorescence radiation differential microscopic imaging device according to claim 1, wherein the beam collimation unit (22) selects a microlens array; the converging lens (23) selects a cylindrical lens.

8. The high-channel fluorescence radiation differential microscopic imaging device according to claim 1, wherein the detection module (6) comprises an optical fiber array end face (31), a detection optical fiber array and a detector array, and multiple fluorescent signals received by the optical fiber array end face (31) are respectively led into the detector array through the detection optical fiber array to be converted into multiple electrical signals.

9. The high-channel fluorescence radiation differential microscopic imaging device according to claim 1, wherein the control unit (7) is used for controlling the optical fiber mode selection module (3) to work, sending an excitation light transmission channel for instructing to gate solid spot excitation and hollow spot excitation to the optical fiber mode selection module (3), and controlling the imaging sequence corresponding to the solid spot excitation and the hollow spot excitation;

when the micro imaging module (5) is controlled by the control unit (7) to work, a scanning instruction is sent to a scanning unit of the micro imaging module (5), and the linear excitation light spot array (29) is controlled to perform two-dimensional/three-dimensional scanning on a sample;

when the control unit (7) collects the electric signals of the detection module (6) and performs super-resolution imaging, the super-resolution microscopic image of the sample is reconstructed and displayed by utilizing a fluorescence radiation differential technology after the solid spot excitation sample image and the hollow spot excitation sample image which are obtained based on the electric signals are obtained according to a scanning time sequence.

10. A method of high-channel fluorescence radiation differential microscopy imaging, characterized in that it employs an apparatus according to any one of claims 1-9, comprising the steps of:

-providing at least one excitation beam (8) of a central wavelength by means of a laser (1);

dividing an excitation beam (8) into excitation sub-beams (9) and (10) by a beam splitter (2) and guiding the excitation sub-beams into two excitation light transmission channels, namely solid spot excitation and hollow spot excitation, of the optical fiber mode selection module (3);

the optical fiber mode selection module (3) is used for time-sharing gating the two excitation light transmission channels, and a fiber array is used for outputting multiple independent solid spot excitation light beams and hollow spot excitation light beams;

the optical field adjustment module (4) is used for carrying out optical field adjustment on the multipath light beams output by the optical fiber mode selection module (3) so as to form a linear excitation light spot array; and is also used for imaging the multipath fluorescence signals obtained from the microscopic imaging module (5) to the detection module (6);

imaging the linear excitation light spot array to a sample to be detected through a microscopic imaging module (5), and collecting fluorescent signals generated by excitation of the sample;

the detector array contained by the detection module (6) receives multiple paths of fluorescent signals in parallel and converts the fluorescent signals into electric signals;

the optical fiber mode selection module (3) and the microscopic imaging module (5) are controlled to work through the control unit (7), and the electrical signals of the detection module (6) are collected and super-resolution imaging is carried out.

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