Multi-focus scanning fluorescence differential microscopic imaging system and imaging method

Abstract

Disclosed in the present invention are a multi-focus scanning fluorescence differential microscopic imaging system and an imaging method, the imaging system comprising: a laser modulation module for generating circularly polarized light, a multi-focus module for converting the circularly polarized light into a parallel beam array, a spatial light modulator for generating a solid beam array or a hollow beam array, a scanning module for exciting a fluorescence signal of a sample, an imaging module for imaging the fluorescence signal, and a control module for controlling the spatial light modulator and the scanning module and performing data processing and image display. Also disclosed is the imaging method using the imaging system. The present invention has the advantages of achieving improvement over the single-point scanning imaging mode of conventional fluorescence differential microscopes, greatly increasing the speed of fluorescence differential microscopic imaging, achieving higher alignment accuracy, and allowing flexible control over the number of hollow beam arrays generated; theoretically, when an N×N point array is used to scan a sample, the final imaging speed of the system is N2 times that of the conventional fluorescence differential microscopes.

Description

A multi-focus scanning fluorescence differential microscopy system and imaging method TECHNICAL FIELD

[0001] The present application relates to a fluorescence differential microscopy system and imaging method, in particular to a multi-focus scanning fluorescence differential microscopy system and imaging method. BACKGROUND

[0002] Microscopes are important scientific tools for human beings to explore the microcosm. The diffraction limit of a microscope in the visible light band is about 200 nm. The objective existence of the diffraction limit hinders the further exploration of the fine life structure by human beings. In recent years, a series of super-resolution microscopy imaging techniques have been proposed to break through the diffraction limit in the scientific community. Among them, the point scanning-based fluorescence differential microscope can achieve a quarter of the illumination wavelength resolution: by using a solid illumination spot and a hollow illumination spot to illuminate the sample in turn, and then multiplying the obtained images by a certain coefficient to perform difference, a super-resolution image can be obtained. The principle is to subtract the point spread function PSF of the solid illumination spot from the point spread function PSF of the hollow illumination spot, so as to obtain a smaller system point spread function PSF. The smaller the system point spread function PSF is, the higher the system imaging resolution is.

[0003] However, the scanning speed of fluorescence differential microscopy is relatively slow, which limits the application range of this technology. Fluorescence differential technology is based on point scanning confocal technology, which uses solid and hollow light spots to illuminate the sample in sequence, and completes the imaging through back-end algorithm (see reference [1] You S, Kuang C, Rong Z, et al. Isotropic superresolution imaging for fluorescence emission difference microscopy[J]. Applied Optics, 2014, 53(33): 7838-7844. [2] Zhu D, Liu W, Zhang Z, et al. Enhancement of fluorescence emission difference microscopy using conjugated vortex phase modulation[J]. Journal of Microscopy, 2018, 272(2): 151-159.). In the imaging process, point scanning and two-beam switching imaging limit the imaging speed of fluorescence differential microscopy, which makes fluorescence differential microscopy unable to meet the needs of real-time imaging of live cells. Therefore, it is urgent to study new methods to improve the imaging speed of fluorescence differential microscopy. Compared to single-point scanning fluorescence differential microscopy, this invention, by employing a multi-focal beam array scanning imaging, can significantly improve the imaging speed of fluorescence differential microscopy while maintaining the resolution of the technique. Theoretically, it can achieve N times the imaging speed of traditional fluorescence differential microscopy. 2 The multi-fold increase (N depends on the multifocal beam array) is beneficial for promoting fluorescence differential microscopy technology and enabling higher-speed super-resolution imaging of living cells and even nerve cells. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a multifocal scanning fluorescence differential microscopy imaging system and imaging method that can significantly improve the imaging speed of fluorescence differential microscopy.

[0005] One of the technical solutions adopted by the present application to solve the above technical problems is: a multi-focus scanning fluorescence differential microscopic imaging system, comprising: a laser modulation module for generating circularly polarized light; a multi-focus module for converting the circularly polarized light into a parallel light beam array; a spatial light modulator for modulating the parallel light beam array to generate a solid light beam array or a hollow light beam array; a scanning module for multi-focus scanning of a sample to excite a fluorescence signal of the sample; an imaging module for imaging the fluorescence signal excited by the sample; and a control module for controlling the vortex phase array loaded into the spatial light modulator to realize switching between the solid light beam array and the hollow light beam array, controlling the scanning timing of the scanning module, and processing and displaying images of the data received by the imaging module.

[0006] Compared with the prior art, the present application has the advantages that the single-point scanning imaging mode of the traditional fluorescence differential microscope is improved, multi-focus scanning imaging is realized by using a multi-focus module and a scanning module, the speed of fluorescence differential microscopic imaging is greatly improved, the method of loading a vortex phase array into a spatial light modulator is used to generate a hollow light beam array, the alignment accuracy is higher, and the number of generated hollow light beam arrays can be flexibly controlled; in theory, if a sample is scanned by using an N×N dot array, the final system imaging speed is N times that of the traditional fluorescence differential microscope. 2

[0007] Preferably, the laser modulation module is composed of a multi-wavelength laser, a multi-mode optical fiber, a collimating lens group, and a laser polarization state modulation element, the multi-wavelength laser outputs multi-wavelength laser which is collimated into a Gaussian-type parallel light by the multi-mode optical fiber and the collimating lens group, and the collimated light beam becomes circularly polarized light by the laser polarization state modulation element.

[0008] Preferably, a light homogenizing element is arranged between the collimating lens group and the laser polarization state modulation element, for shaping the Gaussian-type incident light beam into a uniformly distributed flat-top light beam.

[0009] Preferably, the multi-focus module is composed of a first microlens array, a pinhole array, and a second microlens array, each of which has the same number of rows and columns N, N being a positive integer greater than 1, the first microlens array generates an N×N converging light beam array, the focal points of which are located in a first plane, the pinhole array is arranged on the first plane for filtering non-focal plane stray light, and the second microlens array generates an N×N parallel light beam array. The pinhole array arranged on the first plane can filter the non-focal plane stray light of the first microlens array.

[0010] Preferably, the first microlens array and the second microlens array are symmetrically placed relative to the pinhole array along the light propagation direction. This structure can reduce aberration. ​

[0011] When the control module loads the vortex phase array to the spatial light modulator according to the phase map loading timing, a hollow light beam array is generated, and when the control module does not load the vortex phase array to the spatial light modulator, a solid light beam array is generated.

[0012] Preferably, a dichroic mirror is arranged behind the spatial light modulator, and the light beam emitted by the spatial light modulator is incident on the scanning module through the dichroic mirror to excite the fluorescence signal of the sample by multi-focus scanning of the sample, and the scanning module is arranged to guide the excited fluorescence signal of the sample to the imaging module through the dichroic mirror.

[0013] Preferably, the scanning module comprises a two-dimensional galvanometer group, a scanning lens, a tube lens and an objective lens, the two-dimensional galvanometer group is controlled by the control module to scan the timing to achieve uniform two-dimensional point scanning, and the light beam emitted by the dichroic mirror passes through the two-dimensional galvanometer group, is converged into a light beam array by the scanning lens, is collimated into a parallel light beam array by the tube lens, and is incident on the objective lens, the objective lens converges the light beam array on the sample to form a focal point array, and multi-focus scanning of the sample is achieved, the excited fluorescence signal of the sample is collected by the objective lens, is collimated into a parallel light beam array by the tube lens and the scanning lens, and is incident on the imaging module through the two-dimensional galvanometer group and the dichroic mirror.

[0014] Preferably, the two-dimensional galvanometer group comprises a first galvanometer for scanning the X-axis, a second galvanometer for compensating for scanning distortion of the Y-axis, and a third galvanometer for scanning the Y-axis.

[0015] The second technical solution adopted by the present application to solve the above technical problems is a method for realizing multi-focus scanning fluorescence differential microscopy by using the above imaging system, comprising: the laser modulation module generates circularly polarized light; the multi-focus module converts the circularly polarized light into a parallel light beam array; the parallel light beam array is incident on the spatial light modulator, the spatial light modulator is adjusted by the control module to load the phase map in timing to generate a solid light beam array or a hollow light beam array, the solid light beam array or the hollow light beam array is scanned on the sample by the scanning module to excite the fluorescence signal of the sample; the imaging module images the fluorescence signal excited by the sample; the control module processes the data received by the imaging module: the fluorescence intensity signal of the solid light beam array scanning imaging is defined as I1(x, y), the fluorescence intensity signal of the hollow light beam array scanning imaging is defined as I2(x, y), and the fluorescence signal of the fluorescence differential microscopy system is obtained as I(x, y) = I1(x, y) - m x I2(x, y), wherein m is a coefficient with a value of 0.1-1.0. BRIEF DESCRIPTION OF DRAWINGS

[0016] Fig. 1 is a structural schematic diagram of a multi-focus scanning fluorescence differential microscopic imaging system according to an embodiment of the present application; Fig. 2 is a two-dimensional intensity distribution diagram of a 10x10 solid beam array provided by the embodiment of the present application; Fig. 3 is a two-dimensional intensity distribution diagram of a 10x10 hollow beam array provided by the embodiment of the present application; Fig. 4 is a two-dimensional intensity distribution diagram of a 10x10 solid beam array minus a 10x10 hollow beam array provided by the embodiment of the present application; Fig. 5 is a comparison diagram of normalized curves of a solid beam PSF, a hollow beam PSF and a fluorescence differential PSF provided by the embodiment of the present application; Fig. 6 is a structural schematic diagram of a multi-focus scanning fluorescence differential microscopic ranging provided by an embodiment of the present application. DETAILED DESCRIPTION

[0017] The present application will be further described below in conjunction with the embodiments of the drawings.

[0018] To make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme of the embodiments of the present application will be clearly and completely described below in conjunction with the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0019] Embodiment 1: Fig. 1 is a structural schematic diagram of a multi-focus scanning fluorescence differential microscopic imaging system according to the present embodiment, which comprises a multi-wavelength laser 1, a multi-mode optical fiber 2, a collimating lens group 3, a homogenizer 4, a polarizer 5, a quarter-wave plate 6, a first mirror 7, a 10x10 first microlens array 8, a 10x10 pinhole array 9, a 10x10 second microlens array 10, a second mirror 11, a spatial light modulator 12, a third mirror 13, a dichroic mirror 14, a first galvanometer 15, a second galvanometer 16, a third galvanometer 17, a scanning lens 18, a fourth mirror 19, a tube lens 20, an objective lens 21, a sample stage 22, an imaging lens 23, a CCD camera 24 and a computer 25.

[0020] The multi-focus scanning fluorescence differential microscopic imaging system as shown in Figure 1 is used, and the imaging process is as follows: the laser emitted by the multi-wavelength laser 1 is collimated into a Gaussian parallel light by the multi-mode optical fiber 2 and the collimating lens group 3, the collimated Gaussian parallel light is shaped into a flat-top light beam with uniform light intensity distribution by the homogenizer 4, the flat-top light beam becomes linearly polarized light by the polarizer 5, the linearly polarized light becomes circularly polarized light by the quarter-wave plate 6, the circularly polarized light is incident on the 10x10 first microlens array 8 through the first reflecting mirror 7 and generates a 10x10 converging light beam array, the focal points of the converging light beam array are in the same plane, and are located in the same plane as the 10x10 pinhole array 9, the pinhole array 9 filters out the non-focal plane stray light at the first microlens array 8, the converging light beam array is collimated into a 10x10 parallel light beam array by the 10x10 second microlens array 10, the first microlens array 8 and the second microlens array 10 are symmetrically placed relative to the pinhole array 9 along the light propagation direction, which can reduce aberration; the parallel light beam array is incident on the spatial light modulator 12 through the second reflecting mirror 11, the spatial light modulator 12 is adjusted by the computer 25 to load phase patterns in time sequence to generate a solid light beam array or a hollow light beam array, the solid light beam array or the hollow light beam array is incident on the three-vibration mirror scanning system through the third reflecting mirror 13 and the dichroic mirror 14, the first vibration mirror 15 is used to realize scanning in the X-axis, the second vibration mirror 16 is used to compensate for the "pillow-shaped" distortion of the Y-axis vibration mirror scanning, and the third vibration mirror 17 is used to realize scanning in the Y-axis, so that the three-vibration mirror scanning system can realize relatively uniform two-dimensional point scanning, the light beam is incident on the scanning lens 18 through the third vibration mirror 17, the converging light beam array is collimated into a parallel light beam array by the fourth reflecting mirror 19 and the tube lens 20 and is incident on the objective lens 21, the objective lens 21 converges the light beam array on the sample placed on the sample stage 22 to form a 10x10 focal point array, realizing multi-focus scanning of the sample, the fluorescence signal excited is collected by the objective lens 21 in the original path, collimated into a parallel light beam array by the tube lens 20, the fourth reflecting mirror 19 and the scanning lens 18, transmitted out by the dichroic mirror 14 through the three-vibration mirror scanning system: the third vibration mirror 17, the second vibration mirror 16 and the first vibration mirror 15, converged into a focal point array by the imaging lens 23, and imaged by the CCD camera 24, and data processing and image display are completed by the rear-end computer 25. All optical elements of the imaging system in the example require optical coaxiality. The computer 25 needs to control the spatial light modulator 12 to load the vortex phase array pattern to generate a hollow light beam array, and the computer also needs to control the three-vibration mirror scanning system to complete scanning imaging.

[0021] The fluorescence intensity signal of the solid beam array scanning imaging is I1(x, y), the fluorescence intensity signal of the hollow beam array scanning imaging is I2(x, y), the fluorescence signal of the fluorescence differential microscopy imaging system is I(x, y) = I1(x, y) - m x I2(x, y), m is a coefficient, and the value is 0.7. As shown in FIG. 5, the solid line with a plus sign is the point spread function (PSF) curve of a single solid spot, the solid line with a circle is the point spread function (PSF) curve of a single hollow spot, and the dotted line is the point spread function (PSF) curve of the fluorescence differential microscopy imaging system. As can be seen from the figure, the full width at half maximum (FWHM) of the point spread function curve of the fluorescence differential microscopy imaging system is significantly smaller than the other two curves, so the resolution is improved. The 10 x 10 two-dimensional intensity distribution of the solid beam array is shown in FIG. 2, the 10 x 10 two-dimensional intensity distribution of the hollow beam array generated by the spatial light modulator 12 is shown in FIG. 3, and the two-dimensional intensity distribution of the final solid beam array minus the hollow beam array is shown in FIG. 4. As can be seen from FIG. 4, the size of the spot of the two-dimensional intensity distribution after subtraction is significantly smaller than the size of the solid spot in FIG. 2. Compared with single beam scanning imaging, the speed of 10 x 10 beam array scanning imaging is 10 x 10 = 100 times that of single beam scanning imaging under the same scanning area.

[0022] Example Two: FIG. 6 is a structure diagram of the multi-focal scanning fluorescence differential microscopy ranging system of the present embodiment, which comprises an LED light box 1a, a multi-mode optical fiber 2a, a collimating lens group 3a, a light homogenizer 4a, a polarizer 5a, a quarter-wave plate 6a, a first mirror 7a, a 10 x 10 first microlens array 8a, a 10 x 10 pinhole array 9a, a 10 x 10 second microlens array 10a, a second mirror 11a, a spatial light modulator 12a, a third mirror 13a, a first galvanometer 14a, a second galvanometer 15a, a dichroic mirror 16a, a dispersive objective 17a, a sample stage 18a, an imaging lens 19a, a CCD camera 20a, and a computer 21a.

[0023] The working process of the multi-focus scanning fluorescence differential microscopy ranging system shown in FIG. 6 is as follows: the LED light box 1a can adjust the central wavelength (405 nm, 450 nm, 532 nm, 561 nm, 640 nm, 670 nm), the outgoing light beam is collimated into a Gaussian type parallel light by the multi-mode optical fiber 2a and the collimating lens group 3a, is shaped into a uniformly distributed flat-top light beam by the light homogenizer 4a, becomes linearly polarized light by the polarizer 5a, becomes circularly polarized light by the quarter-wave plate 6a, is incident on the 10x10 first microlens array 8a by the first reflecting mirror 7a and generates a 10x10 converging light beam array, the converging light beam array filters out stray light by the pinhole array 9a, and is collimated into a parallel light beam array by the 10x10 second microlens array 10a, the first microlens array 8a and the second microlens array 10a are symmetrically placed relative to the pinhole array 9a along the light propagation direction, which can reduce aberration; the parallel light beam array is incident on the spatial light modulator 12a by the second reflecting mirror 11a, the spatial light modulator 12a is adjusted by the computer 21a to load a phase pattern in time sequence, generating a solid light beam array or a hollow light beam array, the solid light beam array or the hollow light beam array is incident on the two-mirror scanning system (the first galvanometer 14a and the second galvanometer 15a) by the third reflecting mirror 13a, is transmitted to the dispersion objective 17a by the dichroic mirror 16a, and the converging light beam array is formed on the sample placed on the sample stage 18a to form a 10x10 focal point array, realizing multi-focus scanning of the sample, the sample scattered light is collected by the dispersion objective 17a in the original path, is transmitted to the imaging lens 19a by the dichroic mirror 16a, and the converging light beam is focused into a focal point array by the CCD camera 20a, and the data processing and image display are completed by the rear-end computer 21a. The focusing positions of light beams of different wavelengths are different, and the sample depth information can be calculated by the rear-end algorithm according to the focusing positions of light beams of different wavelengths, the depth information of the sample can be obtained by two-dimensional scanning of the sample, and the ranging of the sample is realized. All optical elements of the imaging system of the present example are required to be optically coaxial. The computer 21a needs to control the spatial light modulator 12a to load a vortex phase array pattern to generate a hollow light beam array, and the computer also needs to control the two-mirror system to complete two-dimensional scanning ranging.

[0024] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A multifocal scanning fluorescence differential microscopy imaging system, characterized in that, include: A laser modulation module is used to generate circularly polarized light; Multifocal module, used to convert circularly polarized light into a parallel beam array; Spatial light modulator, used to modulate a parallel beam array to produce a solid beam array or a hollow beam array; The scanning module is used to perform multifocal scanning of the sample to excite the sample's fluorescence signal; The imaging module is used to image the fluorescence signal excited by the sample; The control module is used to control the vortex phase array loaded onto the spatial light modulator to achieve switching between a solid beam array and a hollow beam array, control the scanning timing of the scanning module, and process and display the data received by the imaging module.

2. The multifocal scanning fluorescence differential microscopy imaging system according to claim 1, characterized in that, The laser modulation module consists of a multi-wavelength laser, a multimode fiber, a collimating lens group, and a laser polarization state modulation element. The multi-wavelength laser outputs multi-wavelength laser light, which is collimated into Gaussian parallel light by the multimode fiber and the collimating lens group. The collimated beam is then converted into circularly polarized light by the laser polarization state modulation element.

3. The multifocal scanning fluorescence differential microscopy imaging system according to claim 2, characterized in that, A beam homogenizing element is provided between the collimating lens group and the laser polarization modulation element to shape the Gaussian incident beam into a uniformly distributed flat-top beam.

4. The multifocal scanning fluorescence differential microscopy imaging system according to claim 1, characterized in that, The multifocal module consists of a first microlens array, a pinhole array, and a second microlens array, all having the same number of rows and columns N, where N is a positive integer greater than 1. The first microlens array generates an N×N converging beam array, with the focal point of the converging beam array located in a first plane. The pinhole array is disposed on the first plane to filter out stray light from the defocused plane. The second microlens array generates an N×N parallel beam array.

5. The multifocal scanning fluorescence differential microscopy imaging system according to claim 4, characterized in that, The first microlens array and the second microlens array are symmetrically placed relative to the pinhole array along the light propagation direction.

6. The multifocal scanning fluorescence differential microscopy imaging system according to claim 1, characterized in that, When the control module loads the vortex phase array onto the spatial light modulator according to the phase diagram loading timing, a hollow beam array is generated; when the control module does not load the vortex phase array onto the spatial light modulator, a solid beam array is generated.

7. The multifocal scanning fluorescence differential microscopy imaging system according to claim 1, characterized in that, A dichroic mirror is provided after the spatial light modulator. The light beam emitted from the spatial light modulator is incident on the scanning module through the dichroic mirror to perform multifocal scanning of the sample and excite the fluorescence signal of the sample. The scanning module then incident the excited sample fluorescence signal on the imaging module through the dichroic mirror.

8. The multifocal scanning fluorescence differential microscopy imaging system according to claim 7, characterized in that, The scanning module includes a two-dimensional galvanometer group, a scanning lens, a tube mirror, and an objective lens. The two-dimensional galvanometer group is controlled by the control module to achieve uniform two-dimensional point scanning. The light beam emitted from the dichroic mirror passes through the two-dimensional galvanometer group, is converged by the scanning lens into a beam array, and then collimated by the tube mirror into a parallel beam array that enters the objective lens. The objective lens converges the beam array to form a focal array on the sample, achieving multifocal scanning of the sample. The excited sample fluorescence signal is collected by the objective lens along the original path, collimated by the tube mirror and the scanning lens into a parallel beam array, and then passed through the two-dimensional galvanometer group and the dichroic mirror to enter the imaging module.

9. A multifocal scanning fluorescence differential microscopy imaging system according to claim 8, characterized in that, The two-dimensional galvanometer assembly consists of a first galvanometer that scans the X-axis, a second galvanometer that compensates for Y-axis scanning distortion, and a third galvanometer that scans the Y-axis.

10. An imaging method using the imaging system of claim 1, characterized in that, include: The laser modulation module generates circularly polarized light; The multifocal module transforms circularly polarized light into a parallel beam array; A parallel beam array is incident on the spatial light modulator. The control module adjusts the spatial light modulator to load a phase map in a time sequence, generating a solid beam array or a hollow beam array. The solid beam array or hollow beam array is scanned by the scanning module to excite the fluorescence signal of the sample through multifocal scanning. The imaging module images the fluorescence signal excited by the sample. The control module processes the data received by the imaging module: the fluorescence intensity signal of the solid beam array scanning imaging is defined as I1(x,y), and the fluorescence intensity signal of the hollow beam array scanning imaging is defined as I2(x,y). The fluorescence signal of the fluorescence differential microscopy imaging system is obtained as I(x,y) = I1(x,y) - m × I2(x,y), where m is a coefficient with a value of 0.1-1.0.

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