CN107389631B

CN107389631B - High-speed multicolor multi-modal structured light illumination super-resolution microscopic imaging system and method thereof

Info

Publication number: CN107389631B
Application number: CN201710495353.7A
Authority: CN
Inventors: 李栋; 李迪; 张思微; 刘勇
Original assignee: Institute of Biophysics of CAS
Current assignee: Beijing Nathan Optoelectronic Technology Co ltd
Priority date: 2017-04-28
Filing date: 2017-06-26
Publication date: 2020-07-07
Anticipated expiration: 2037-06-26
Also published as: CN107389631A

Abstract

The high-speed multicolor multimode structured light illumination super-resolution microscopic imaging system is characterized in that laser generated by a multicolor light source is incident on a high-speed gating switching module; the high-speed gating switching module selects single or multiple colors of laser to irradiate the structured light generation and modulation module; the structured light generating and modulating module generates periodically modulated structured light, controls the direction and the phase of the structured light, and transmits the modulated laser to the polarization control module; the polarization control module adjusts the polarization direction of the laser, so that the contrast of the structure illumination stripes is high, and the structure illumination stripes are transmitted to the spatial filtering module; the spatial filtering module filters redundant stray light, and then laser is irradiated onto the sample module through the fluorescence module and the objective lens in sequence; the signal light emitted by the sample module is collected by the objective lens, then the fluorescence module separates the excitation light from the signal light, and finally the signal light is received by the detection module. The structured light illuminating fringe has high contrast and high imaging speed, and simultaneously, the optimal performance can be achieved by at least 5 colors of imaging.

Description

High-speed multicolor multi-modal structured light illumination super-resolution microscopic imaging system and method thereof

Technical Field

The invention relates to the field of microscope imaging, in particular to a high-speed multicolor multi-modal structured light illumination super-resolution microscopic imaging system and method.

Background

Optical microscopy has greatly facilitated the development of biology since the invention of the 17 th century. As early as 1665, humans discovered, described and defined for the first time by means of light microscopy cells, the fundamental unit of this vital activity, and thus initiated the transition of biological studies from macroscopic description to microscopic quantification. It can be said that the progress of optical microscopy, especially fluorescence microscopy, is a very important driving force for the rapid progress of life science research in recent decades. It is well known that the fluctuating nature of light limits the resolution of optical microscopes. As early as over 100 years ago, Abbe (Abbe), a German scientist, demonstrated that the limiting resolution of optical microscopes, typically around 200nm for green fluorescence, cannot be less than half the wavelength of light used, a resolution limit known as the diffraction limit. 200nm is about 100 times larger than the size of a single molecule, which means that optical microscopy cannot track subtle changes in biological processes, resolve the fine structure of organelles, and define the dynamic distribution of specific molecules within organelle substructures. Thus severely limiting the possibility of scientists to find and answer deep life science questions using optical microscopy. Electron microscopy, although it has sub-nanometer level spatial resolution, cannot be used to study living cells. The improvement of the resolution of optical imaging systems is therefore a long-sought goal in the field of optical research and an important direction in the development of new modern optical technologies. In recent 20 years, new technologies for breaking through diffraction limit resolution have been emerging, and a german scientist step hel invented a Stimulated Emission Depletion (STED) microscope technology [1,2], and a repeated saturated Optical fluorescence conversion (RESOLFT) microscope technology popularized based on the STED concept, wherein the principle is to reduce a Point Spread Function (PSF) on a spatial domain to achieve an effect of super resolution. The american scientist Eric Betzig invented light activated positioning microscope (PALM) 3, and the random Optical reconstruction microscope (STORM) invented by professor of china at harvard university 4, both of which are super-resolution Microscopy based on optically controlled single-molecule positioning. The point spread function imaging of the super-resolution imaging technology based on PALM or STORM is still consistent with that of the traditional microscopic imaging, and a super-resolution image is obtained by repeatedly activating and quenching fluorescent molecules, so the time resolution is low. The essence of these two super-resolution imaging techniques is to trade temporal resolution for spatial resolution. And the STED causes photobleaching and even phototoxicity due to overlarge light intensity, and is not suitable for biological living body research.

Among many super-resolution imaging technologies, structured light illumination (SIM) is used as a wide-field microscopic imaging technology, and the principle is that by modifying uniform illumination of kohler illumination into periodically modulated illumination light, high spatial frequency information that cannot be obtained originally is modulated to a low-frequency region, and then passes through a microscope imaging system with a limited frequency band (diffraction limit), and high-frequency components are restored by frequency domain separation and frequency shift decoding, so that a super-resolution image that breaks through the diffraction limit is obtained. Therefore, the imaging speed is faster than that of a STED (step scanning) point scanning imaging mode or PALM (planar imaging) mode and other single-molecule space positioning multi-time imaging combined with a fluorescent molecule switching effect, and the method is more suitable for developing living body, high-speed and low-bleaching biological living body research. Meanwhile, the maximum difference between the structured light and the traditional fluorescence microscope is that exciting light is modulated, so that structured light illumination super-resolution imaging can be realized only by modifying an illumination light path. Moire interference fringes formed by utilizing the frequency characteristics of the structured light and the spatial frequency components of the observed sample can improve the transverse resolution of imaging; meanwhile, the single-cell super-resolution micro-imaging system has better chromatographic capability, can realize the super-resolution rapid three-dimensional imaging of single cells by combining with the super-resolution micro-imaging technology on a transverse plane, and has important scientific significance and application value, and the structured light technology becomes the leading edge and the hot spot of international research in recent years [5-6,10-11 ].

In 1997, Neil et al [7] first performed tomography using structured light illumination, acquired three corresponding initial images by illuminating a sample using three cosine-modulated structured light with different initial phases, and reconstructed a tomographic image using a simple algorithm, improving the longitudinal resolution of the microscope. Around 2000, Heintzmann [8] and Gustafsson [9] et al used the same device as Neil to perform experiments, but used an image reconstruction algorithm different from Neil to improve the lateral resolution of the reconstructed image and pointed out that linear structured light illumination only increased the imaging resolution by a factor of two at most. The linear structured light illumination fluorescence microscope means that the emission light intensity of a fluorescent group and the light intensity of structured illumination light are in a linear relation.

The research in the field of the structured light illumination super-resolution microscope in China starts relatively late, but in recent years, a lot of special work is also done. The main research units at present are the institute of biophysical sciences of the Chinese academy of sciences, the institute of optical precision mechanics of Western Ann, the institute of biomedical engineering and technology of Suzhou, the university of Beijing, the university of science and technology of Huazhong, etc. [10-16 ]. Among them, the microscopic technique based on the digital micromirror device and LED illumination, which is the institute of precision optics and mechanics of west ampere, the academy of sciences of china, is most representative [13 ]. The device adopts the LED light source to reduce the background noise caused by unfavorable factors such as speckle interference and the like, and can obtain the spatial resolution of 90nm and the slice imaging speed of 190 ms. The research group of the university of science and technology in China combines a differential interference phase contrast technology and a structured light illumination technology to realize super-resolution imaging of coherent light [15 ]. However, the same international problems are met, and high-speed, multicolor and multi-mode super-resolution in vivo imaging is still difficult to realize. In addition, the existing structured light microscope is difficult to flexibly select an imaging mode according to imaging requirements and sample characteristics and adjust imaging depth.

At present, commercial structured light illumination super-resolution imaging equipment is only owned by a few foreign companies such as GE, Zeiss and Nikon, the domestic super-resolution imaging equipment is almost blank and is imported, the price is high, and the direction and the phase of structured light are changed by rotating the grating based on the traditional body grating diffraction interference principle, so that mechanical movement is involved, and the response is slow. In addition, the stability and flexibility are poor, and the optimal illumination condition cannot be achieved for different excitation wavelengths. These factors make the current structured light microscope slow in imaging speed, difficult to achieve optimal multicolor imaging performance, and unable to maintain optimal performance in each imaging mode, which is not conducive to its popularization in life science research.

Cited documents:

1.HELL S W,WICHMANN J.Breaking the diffraction resolution limit bystimulated emission:stimulated-emission-depletion fluorescence microscopy[J].Optics letters,1994,19(11):780-2.

2.KLAR T A,HELL S W.Subdiffraction resolution in far-fieldfluorescence microscopy[J].Optics letters,1999,24(14):954-6.

3.BETZIG E,PATTERSON G H,SOUGRAT R,et al.Imaging intracellularfluorescent proteins at nanometer resolution[J].Science,2006,313(5793):1642-5.

4.RUST M J,BATES M,ZHUANG X.Sub-diffraction-limit imaging bystochastic optical reconstruction microscopy(STORM)[J].Nature methods,2006,3(10):793-6.

5.SHAO L,KNER P,REGO E H,et al.Super-resolution 3D microscopy of livewhole cells using structured illumination[J].Nature methods,2011,8(12):1044-6.

6.KNER P,CHHUN B B,GRIFFIS E R,et al.Super-resolution videomicroscopy of live cells by structured illumination[J].Nature methods,2009,6(5):339-42.

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R,WILSON T.Method of obtaining optical sectioningby using structured light in a conventional microscope[J].Optics letters,1997,22(24):1905-7.

8.HEINTZMANN R,CREMER C G.Laterally modulated excitation microscopy:improvement of resolution by using a diffraction grating；proceedings of theBiOS Europe'98,F,1999[C].International Society for Optics and Photonics.

9.GUSTAFSSON M G,AGARD D A,SEDAT J W.Doubling the lateral resolutionof wide-field fluorescence microscopy using structured illumination；proceedings of the BiOS 2000 The International Symposium on BiomedicalOptics,F,2000[C].International Society for Optics and Photonics.

10.LI D,SHAO L,CHEN B-C,et al.Extended-resolution structuredillumination imaging of endocytic and cytoskeletal dynamics[J].Science,2015,349(6251):aab3500.

11.Zhang X,Zhang M,Li D,He W,Peng J,Betzig E,Xu P.Highly photostable,reversibly photoswitchable fluorescent protein with high contrast ratio forlive-cell superresolution microscopy[J].Proceedings of the National Academyof Sciences,2016,Aug 25:201611038.

12.YANG X,TZENG Y-K,ZHU Z,et al.Sub-diffraction imaging of nitrogen-vacancy centers in diamond by stimulated emission depletion and structuredillumination [J].RSC Advances,2014,4(22):11305-10.

13.DAN D,LEI M,YAO B,et al.DMD-based LED-illumination super-resolution and optical sectioning microscopy[J].Scientific reports,2013,3(1116).

14.ZHOU X,LEI M,DAN D,et al.Double-Exposure Optical SectioningStructured Illumination Microscopy Based on Hilbert Transform Reconstruction[J].PloS one,2015,10(3):e0120892.

15.CHEN J,XU Y,LV X,et al.Super-resolution differential interferencecontrast microscopy by structured illumination[J].Optics express,2013,21(1):112-21.

16.LAL A,SHAN C,XI P.Structured illumination microscopy imagereconstruction algorithm[J].2016

disclosure of Invention

The invention provides a high-speed multicolor multimode structured light illumination super-resolution microscopic imaging system and method, aiming at overcoming the defects that the existing structured light illumination microscope system is low in original image acquisition speed, difficult to optimize imaging conditions of exciting light with any wavelength in real time, low in structured light illumination bright fringe contrast, incapable of flexibly switching among different imaging modes and the like.

According to one aspect of the invention, a high-speed multicolor multimode structured light illumination super-resolution microscopic imaging system is provided, which comprises a multicolor light source 1, a high-speed gating switching module 2, a structured light generating and modulating module 3, a polarization control module 4, a spatial filtering module 5, a fluorescence module 6, an objective lens 7 and a detection module 9; the laser generated by the multicolor light source 1 is incident on the high-speed gating switching module 2; the high-speed gating switching module 2 selects single or multiple colors of laser to irradiate the structured light generation and modulation module 3; the structured light generation and modulation module 3 generates periodically modulated structured light and controls the direction and the phase of the structured light, and the modulated laser is transmitted to the polarization control module 4; the polarization control module 4 adjusts the polarization direction of the laser, so that the contrast of the structure illumination stripes is high, and the structure illumination stripes are transmitted to the spatial filtering module 5; the spatial filtering module 5 filters redundant stray light, and then laser sequentially passes through the fluorescence module 6 and the objective lens 7 to irradiate a sample module 8; the signal light emitted from the sample module 8 is collected by the objective lens 7, then the fluorescence module 6 separates the excitation light from the signal light, and finally the signal light is received by the detection module 9.

Further, the structured light generation and modulation module is used for generating at least two beams of coherent or semi-coherent light, and when the beams intersect in the sample, periodic structured light can be generated in an overlapping area for illuminating the sample; the structured light generation and modulation module comprises a cascade beam splitter, a grating, a digital micromirror device, or a spatial light modulator.

Further, the polarization control module is a polarization rotator composed of a variable phase retarder and a quarter-wave plate, or a half-wave plate fixed on the rotating device, and is used for rotating the linear polarization direction of the light source to obtain high-contrast structured light stripes in the light beam overlapping area.

Further, the multicolor light source is a broad spectrum light source, or a plurality of narrow-band single LED light sources, or a plurality of lasers with good monochromaticity.

Further, when a monochromatic laser is adopted, the high-speed gating switching module is an acousto-optic tunable filter or an electro-optic modulator and is used for selecting one or more lasers with specific wavelengths at a high speed to be coupled into a subsequent optical path;

when a broad spectrum light source or a plurality of narrow-band single LED light sources are adopted, the high-speed gating switching module is a high-frequency digital signal generator or a high-speed optical filter turntable.

Further, the spatial filtering module comprises a high-speed vibration motor and a filtering template; the filtering template is installed on the high-speed vibration motor and used for filtering redundant stray light of the structural light stripe in a specific direction.

Further, the fluorescence module comprises a barrel mirror, a dichroic sheet and a filter; the detection module comprises a scientific research camera for detecting signal light and a filter turntable for selecting a proper filter for a specific signal light wavelength range to remove exciting light.

Furthermore, the detection module comprises at least two paths, wherein when one path is used for collecting, the other path can prepare a corresponding optical filter in advance according to the spectral range of the next signal light, and the non-stop collection is realized through alternate operation.

According to another aspect of the present invention, there is provided an imaging method of a high-speed multi-color multi-modal structured light illuminated super-resolution microscopy imaging system, in a dual-beam incident structured light illumination mode forming total internal reflection, grazing incidence or falling incidence, comprising the steps of:

step 10, laser generated by the multicolor light source (1) is incident on the high-speed gating switching module (2), the laser with a specific wavelength selected by the high-speed gating switching module (2) is irradiated on the structured light generation and modulation module (3), and the structured light generation and modulation module (3) generates laser with energy concentrated on positive and negative first-order diffraction beams and weaker high-order diffraction beams; then, the polarization direction of the laser is perpendicular to the connection line direction of the positive and negative first-level through a polarization control module (4), the positive and negative first-level of the structured light is selected through a spatial filtering module (5), and then the structured light passes through a fluorescence module (6) and is placed on a sample on an objective table from the lower part through an objective (7) to form a structured illumination light field;

step 20, controlling the structured light generation and modulation module to load at least one structured illumination light field with the same spatial direction; when more than one structure illumination light field is loaded, the illumination light fields of different structures are relatively translated, namely have different phases;

step 30, correspondingly acquiring two-dimensional images of different phases of the structure illumination light field through a detection module (9); fourier transformation is respectively carried out on the two-dimensional images with different phases, and then each Fourier spectrum component is solved through an image processing algorithm to obtain high-order and low-order Fourier spectrum information of the sample in the direction perpendicular to the current stripe orientation;

step 40, controlling the structural light generation and modulation module to change the spatial direction of the N times of structural illumination light fields, so that the spatial direction included angle of two adjacent structural light fields is 180/N degrees; repeating step 30 to obtain high order and low order fourier spectral information of the sample in a spatial direction 180/N ° apart from the spatial direction in step (20);

step 50, moving the high-order and low-order Fourier spectrum information of the samples in the N spatial directions obtained in the step 30 and the step 40 to the correct positions in the sample spectrums where the high-order and low-order Fourier spectrum information are located, digitally filtering each frequency spectrum component, and then superposing the frequency spectrum components together according to a certain weight to further obtain all the high-order and low-order Fourier spectrum information of the samples; the N > is 2;

and step 60, performing inverse Fourier transform on all high-order and low-order Fourier spectrum information of the sample, and performing deconvolution operation to obtain a super-resolution sample image.

According to another aspect of the present invention, there is provided an imaging method of a high-speed multi-color multi-modal structured light illuminated super-resolution microscopy imaging system, in an epi-structured light illumination mode formed by three beams of light incidence, comprising the steps of:

step 10, laser generated by the multicolor light source (1) is incident on the high-speed gating switching module (2), the laser with specific wavelength selected by the high-speed gating switching module (2) is irradiated on the structured light generation and modulation module (3), and the structured light generation and modulation module (3) generates laser with energy concentrated on zero-order and positive and negative first-order diffraction beams and weaker high-order diffraction beams; then, the polarization direction of the laser is perpendicular to the connection line direction of the positive and negative first-level through a polarization control module (4), the positive and negative first-level of the structured light is selected through a spatial filtering module (5), and then the structured light passes through a fluorescence module (6) and is placed on a sample on an objective table from the lower part through an objective (7) to form a structured illumination light field;

step 20, controlling the structured light generation and modulation module to load at least one structured illumination light field with the same spatial direction in sequence; when more than one structure illumination light field is loaded, the illumination light fields of different structures are relatively translated, namely have different phases;

step 40, controlling the structural light generation and modulation module to change the spatial direction of the N times of structural illumination light fields, so that the spatial direction included angle of two adjacent structural light fields is 180/N degrees; repeating step 30 to obtain high order and low order fourier spectral information of the sample in a spatial direction 180/N ° apart from the spatial direction in step 20;

step 50, moving the sample along the direction of the optical axis to enable the focal plane of the objective lens to coincide with sample planes of different depths, and repeating the steps 10 to 40 to obtain high-order and low-order Fourier spectrum information of the sample in the N spatial directions of the different sample planes;

step 60, moving the high-order and low-order Fourier spectrum information of the samples in the N spatial directions of the different sample surfaces obtained in the step 50 to the correct positions in the sample spectrums, digitally filtering each spectrum component, and then superposing the spectrum components together according to a certain weight to further obtain all the high-order and low-order Fourier spectrum information of the samples;

step 60, performing inverse Fourier transform on all high-order and low-order Fourier spectrum information of the sample, and performing deconvolution operation at the same time to obtain a super-resolution sample image; and N > is 2.

The high-speed multicolor multimode structured light illumination super-resolution microscopic imaging system and the method provided by the invention achieve high-speed accurate control on the period, direction, phase and polarization direction of the structured light illumination stripes by optimizing the response of each module and the overall system control, and can realize the optimized structured light illumination conditions under a plurality of excitation wavelengths and different imaging modes. And obtaining super-resolution sample information from the acquired original image through a frequency shift image reconstruction algorithm. The invention has the advantages of high contrast ratio of the structured light illumination stripes, high imaging speed, and capability of reaching about 100 high-resolution images per second, and simultaneously, the optimal performance of at least 5-color imaging can be achieved, thus being more suitable for real-time dynamic observation of biological living body cells.

Drawings

FIG. 1 is a block diagram of a high-speed multi-color multi-modal structured light illuminated super-resolution microscopy imaging system according to an embodiment of the present invention;

FIG. 2 is a block diagram of a multi-color gating module formed by multiple lasers with good monochromaticity and AOTF according to an embodiment of the present invention;

FIG. 3 is a block diagram of a multi-color high-speed gating module using a broad-spectrum light source and a high-speed filter switching wheel according to an embodiment of the present invention;

FIG. 4 is a block diagram of a multi-color high-speed gating module using a plurality of narrow-band high-brightness LEDs and a high-speed filter switching wheel according to an embodiment of the present invention;

FIG. 5 is a schematic diagram of a module formed by a Spatial Light Modulator (SLM) according to an embodiment of the present invention;

FIG. 6 is a block diagram of a Digital Micromirror Device (DMD) according to an embodiment of the present invention;

FIG. 7 is a schematic overall structure diagram of a preferred embodiment of the high-speed multi-color multi-modal structured light illuminated super-resolution microscopy imaging system provided by the present invention;

FIGS. 8 a-8 d are schematic diagrams of four modes of operation of a preferred embodiment of the present invention, provided by an example of the present invention;

FIG. 9 is a graph of the intensity of the interference light of S-S polarized and P-P polarized beams with different NA along the x-axis.

Detailed Description

Referring to fig. 1, an embodiment of the present invention provides a high-speed multicolor multi-modal structured light illumination super-resolution microscopic imaging system, which includes a multicolor light source 1, a high-speed gating switching module 2, a structured light generating and modulating module 3, a polarization control module 4, a spatial filtering module 5, a fluorescence module 6, an objective lens 7, a sample module 8, and a detection module 9. Laser generated by the multicolor light source 1 is incident on the high-speed gating switching module 2; the high-speed gating switching module 2 selects one or more colors of laser to irradiate the structured light generation and modulation module 3; the structured light generating and modulating module 3 generates periodically modulated structured light and accurately controls the direction and the phase of the structured light, the modulated laser is transmitted to the polarization control module 4, and the polarization direction of the laser is adjusted by the polarization control module 4 to obtain structured light with high stripe contrast; the polarized and adjusted light passes through the spatial filtering module 5, redundant stray light is filtered, then the stray light passes through the fluorescence module 6, the objective lens 7 projects to the sample module 8, and fluorescence emitted by the sample is received by the detection module 9 through the objective lens 7 and the fluorescence module 6.

FIG. 2 is a block diagram of a multi-color high-speed gating module composed of a multi-channel laser with good monochromaticity and AOTF, wherein the laser is used for activating fluorescent proteins, such as a 405nm continuous solid laser, and also can be used for exciting the fluorescent proteins to emit fluorescence, such as 445nm, 488nm, 515nm, 560nm, 642nm, etc. The embodiment is specifically a five-path laser, and after laser is emitted, the laser is expanded into uniform parallel beams with the same diameter through two corresponding short-focus lens groups, and then the beams are integrated into one beam by using corresponding dichroic filters. And lasers can be added continuously, and each laser needs to be added with a corresponding lens group and a dichroic plate.

Fig. 3 is a block diagram of a multi-color high-speed gating module using a broad-spectrum light source and a high-speed filter switching wheel, wherein the broad-spectrum light source is a high-power mercury lamp, xenon lamp, halogen lamp, and the like. The high-speed optical filter switching wheel is used for switching the optical filter at a high speed to obtain the required uniform light illumination with specific wavelength.

FIG. 4 is a multi-color high-speed gating module diagram composed of a plurality of narrow-band high-brightness LEDs and a high-speed filter switching wheel, wherein the high-brightness LEDs cover light bands from near ultraviolet to near infrared through a plurality of band combinations, a high-frequency digital signal generator is used for controlling the on and off of one or more LEDs, and if excitation light with better monochromaticity is desired, the narrow-band filters can be switched at high speed by the high-speed filter switching wheel, so that the monochromaticity of the corresponding LEDs is optimized.

Fig. 5 is a schematic diagram of a module constructed by using a Spatial Light Modulator (SLM) as a main body, and is an embodiment of a specific implementation of a structured light generation and regulation module, a polarization control module, and a spatial filtering module. The light after beam expansion and collimation is incident on a Polarization Beam Splitter (PBS), wherein the projection of p-polarized light can not enter a light path, the reflection of s-polarized light is incident on a silicon-based liquid crystal chip SLM through a half-wave plate and modulated by a loaded specific pattern, and then the polarization state of exciting light is adjusted through a polarization rotator (polarization rotator) to be always vertical to an incident plane formed by positive and negative first-order diffraction orders, namely the s-polarized state is achieved. Then, a specific order of light enters a subsequent light path by using a motor-driven switcher (Galvo) in cooperation with a specific mask.

Fig. 6 is a schematic diagram of a module formed by a Digital Micromirror Device (DMD), which is an embodiment of a structured light generation and regulation module, a polarization control module, and a spatial filtering module. The collimated light irradiates the DMD at a specific angle, and the light beam is modulated by the DMD and then emitted to the quarter-wave plate, so that the light is changed from linear polarization to circular polarization.

FIG. 7 is a schematic diagram of the overall structure of a preferred embodiment of the high-speed multi-color multi-modal structured light illumination super-resolution micro-imaging system according to the present invention. Five lasers with different wavelengths and good monochromaticity are expanded into uniform parallel beams with the same diameter (such as 1.7mm) through a beam expanding system consisting of two short-focus lenses. Five lasers were integrated into a common line using five different dichroic patches. The power of either laser is then modulated at high speed (greater than kilohertz) using either an acousto-optic modulator (AOTF) or electro-optic modulator (EOM). The integrated exciting light passes through a lens 11, a lens 12 expands to 15mm and then enters a Polarization Beam Splitter (PBS), wherein p-polarized light cannot enter a light path in a projection mode, s-polarized light is reflected and enters a silicon-based liquid crystal chip (SLM) through a half-wave plate and is modulated through a loaded specific pattern, then the polarization state of the exciting light is adjusted through a polarization rotator (Galvo), a motor-driven switcher (Galvo) is used for being matched with a mask plate to filter out useless high-level diffraction, an objective lens is finally introduced, the period of a grating pattern on the SLM is accurately controlled by utilizing a fringe generating algorithm, so that the Numerical Aperture (NA) of the incident light is adjusted, and at the moment, the four modes can be divided into four modes according to the relation between the NA of the incident: 1. when the incident light NA is greater than the total reflection critical NA, the incident light is totally reflected at the interface between the glass and the sample as shown in FIG. 8(a), so that a totally reflected evanescent wave light field is formed at the interface between the glass slide and the sample, the illumination depth is 50-150 nm, and the light intensity is reduced along with the depth index. The two incident plane waves interfere to form a spatially sinusoidally modulated interference fringe to excite a fluorescent signal within the sample. This enables Total Internal reflection structured light illumination microscopy, TIRF-SIM (Total Internal reflection microscopy-SIM) mode. 2. When the incident light NA is slightly smaller than the total reflection critical NA, the incident light grazes and enters to form a light sheet with uniform light intensity parallel to the cover glass as shown in FIG. 8(b), and the thickness is 700-1000 nm. Namely Grazing Incidence structured light illumination microscopic imaging, the GI-SIM (GI-SIM) mode can obtain an image with high contrast and high signal-to-noise ratio similar to the TIRF-SIM, and meanwhile, the imaging depth is greatly increased, and the light intensity is not exponentially attenuated along with the depth. 3. When the incident light NA is smaller than the total reflection critical NA, as shown in fig. 8(c), the structured light illumination imaging is performed by the large-inclination corner emission illumination, and the HI-SIM (HI-SIM) mode can perform structured light illumination imaging on any depth plane in the sample, and can synthesize a three-dimensional super-resolution image after imaging at different depths. 4. As shown in fig. 8(D), the three-dimensional structured light imaging mode, 3D-SIM, (three dimensional SIM, 3D-SIM) uses zero order and positive and negative first order interference of diffraction order to form three-dimensional modulated structured light fringes, and by performing structured light illumination on sample planes of different depths and collecting original images, resolution can be uniformly improved in a three-dimensional space. The light fields with the four spatial structures illuminate a biological sample with a fluorescent mark, fluorescence emitted by the sample is collected by an objective lens and is subjected to signal acquisition by using a high-sensitivity detector, in order to improve imaging speed and reduce mutual interference among fluorescent signals with different wavelengths, a dichroic mirror is used for splitting the fluorescent signals, and then the two detectors are used for acquiring the fluorescent signals with different wavelengths. And performing data reconstruction on the acquired image to obtain a super-resolution image.

The specific implementation of this embodiment of the invention is described below with reference to fig. 7:

1. the sample is placed in the sample chamber, the sample plane is found through focusing, and the imaging surface of the microscope is always kept at the corresponding sample plane by using the automatic focusing function.

2. The traditional microscopic uniform illumination is adopted and the fluorescence of the sample is excited, a color filter is used for separating the reflected excitation light from the fluorescence signal, an ocular lens is used for observing the fluorescence signal of the sample, and the interested region is found by moving a displacement platform for super-resolution imaging.

3. Before super-resolution imaging, in order to achieve the purpose of high-speed acquisition, required structured light pattern data with different wavelengths and different numerical apertures are downloaded into an on-board cache of an SLM (selective laser modulation), and a corresponding area is defined in a memory in advance for storing acquired fluorescent pictures.

4. When super-resolution imaging is carried out, a first structured light pattern is uploaded to an SLM, the working frequency of the AOTF is controlled at the same time, so that specific exciting light penetrates through the AOTF and irradiates on the SLM to form required structured light and illuminate a sample, and a high-speed high-quantum-efficiency detector is used for collecting a fluorescence signal emitted by the sample to obtain a fluorescence signal photo corresponding to the first structured light. And after the fluorescent picture corresponding to the first structural picture is acquired, loading the second phase picture on the SLM and repeating the shooting process until the original images required by the corresponding imaging modes are acquired, and then completing the shooting process of a super-resolution image. In order to effectively remove interference generated by high-grade diffraction light spots of the SLM, the voltage of the electric mask plate needs to be adjusted once when the direction of the SLM phase diagram is changed once, so that the corresponding required diffraction order can pass through the mask plate. In the whole process, the FPGA is used for accurately controlling the switching time of the picture sequence on the SLM, the light transmission time of the AOTF, the exposure time of the detector and the like, and the time accuracy can reach microsecond mu s magnitude.

5. And carrying out image reconstruction on the acquired original image data at the graphic workstation. Thereby obtaining a super-resolution imaging picture.

Fig. 8 is a schematic diagram of four modes of operation of the preferred embodiment of the present invention. As shown in fig. 8(a), incident light is totally reflected at the glass interface, the illumination depth is 50-150 nm, the light intensity is exponentially decreased with the depth, so that a totally internally reflected evanescent wave light field is formed on the surface of the glass slide, and a required stripe-type space light field with sinusoidal variation, i.e., a TIRF-SIM mode, is formed by means of laser interference. 2. When the incident light NA is slightly smaller than the total reflection critical NA, the incident light grazes and enters to form a light sheet with uniform light intensity parallel to the cover glass as shown in FIG. 8(b), and the thickness is 700-1000 nm. I.e. GI-SIM mode, a high contrast and high signal-to-noise ratio image similar to TIRF-SIM can be obtained, while the imaging depth is greatly increased. 3. When the incident light NA is smaller than the total reflection critical NA, as shown in fig. 8(c), the illumination imaging mode with the large-tilt-angle illumination structure is adopted, and the background signal can be effectively removed and the optical resolution can be improved to a certain extent under the condition of optical layer cutting. FIG. 8(d) shows three light beams incident to form a three-dimensional structured light illumination imaging mode, which can uniformly improve the three-dimensional optical resolution.

FIG. 9 is a graph showing the variation of the interference intensity along the x-axis between S-S polarized and P-P polarized beams of objective lenses with different NA at an incident wavelength of 488 nm. As shown in FIG. 9, S, P represents two orthogonal linear polarizations, and θ is the angle between the incident light of the linearly polarized light beam and the Z-axis. It can be seen that the contrast of the interference fringes is always 1 regardless of any included angle theta in the S-S linearly polarized light beam interference, and the contrast of the interference fringes of the P-P linearly polarized light beam changes along with the change of theta. Whereas the S-P mode does not produce interference fringes (not shown).

The high-speed multicolor multimode structured light illumination super-resolution microscopic imaging system provided by the invention has the following beneficial effects:

1. the system can provide multicolor imaging by utilizing a broad-spectrum light source or a combination of a plurality of narrow-band high-brightness LEDs and a high-speed filter switching rotating wheel, and also can provide high-speed multicolor illumination by adopting a plurality of lasers with good monochromaticity in combination with AOTF or EOM.

2. The structured light generation and modulation module is used for modulating the illumination light into the structured light, in order to reconstruct a uniform super-resolution picture in each direction, the structured light needs to be subjected to stripe illumination in at least two directions, and each direction is at least illuminated by one phase of one structured light stripe. The key point of obtaining a high-quality super-resolution image by the structured light illumination super-resolution microscopic imaging technology is to ensure that the contrast of the stripes can reach or approach 100% when the stripes rotate to any direction, so that the signal intensity of high-resolution information can approach the theoretical maximum value, and a super-resolution image with a high signal-to-noise ratio is finally obtained. The polarization control module is used for rotating the polarization direction of incident light while the illumination fringes rotate, so that the two plane waves can be completely interfered to generate high-contrast fringes under any space orientation condition.

3. Unnecessary diffraction orders are filtered by utilizing a spatial filtering module, and a mask plate is installed on an electric switcher (Galvo motor) to realize high-speed rotation and switching to a specific position, so that diffraction order light for generating structural light in a corresponding direction can pass through the mask plate, and the rest stray diffraction order light is filtered.

4. And the obtained high-contrast structured light stripe light is projected to a sample after being reduced by using the fluorescence module and the objective lens, a fluorescence signal in the sample is excited, and the acquired information is imaged on a detection system by using the objective lens and the fluorescence module. The sample chamber is used for maintaining the living physiological condition of the living sample, and the rapid three-dimensional collection of the sample can be realized by using the rapid high-precision three-dimensional displacement platform. By using the multi-channel detector, one channel of acquisition and one channel of preparation are carried out during multicolor imaging, and the acquisition speed can be further improved by alternate acquisition.

5. The invention needs each part of the whole system to work coordinately with a specific time sequence, each module must be implemented with accurate logic control, and after the logic is definitely controlled, the FPGA program is compiled to make the control card work independently, and generate various synchronous signals to control the high-speed multicolor structure light illumination super-resolution micro-imaging system in real time.

The above description is only for the preferred embodiment of the present invention and is not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the present specification and the drawings, or any other related technical fields, are included in the scope of the present invention.

Claims

1. A high-speed polychrome multi-modal structured light illumination super-resolution microscopic imaging system which characterized in that: the system adopts a total internal reflection illumination mode formed by double-beam incidence, a grazing incidence or falling structured light illumination mode or a falling structured light illumination mode formed by three-beam incidence, and comprises a multicolor light source (1), a high-speed gating switching module (2), a structured light generating and modulating module (3), a polarization control module (4), a spatial filtering module (5), a fluorescence module (6), an objective lens (7) and a detection module (9); laser generated by the multicolor light source (1) is incident on the high-speed gating switching module (2); the high-speed gating switching module (2) selects single or multiple colors of laser to irradiate the structured light generation and modulation module (3); the structured light generation and modulation module (3) generates structured light which is periodically modulated, controls the direction and the phase of the structured light, and transmits the modulated laser to the polarization control module (4); the polarization control module (4) adjusts the polarization direction of the laser, so that the contrast of the structure light illumination stripes is high, and the structure light illumination stripes are transmitted to the spatial filtering module (5); the spatial filtering module (5) filters redundant stray light, and then laser sequentially passes through the fluorescence module (6) and the objective lens (7) to irradiate a sample module (8); the signal light emitted by the sample module (8) is collected by the objective lens (7), then the fluorescence module (6) separates the excitation light from the signal light, and finally the signal light is received by the detection module (9); the multicolor light source is a broad spectrum light source, or a plurality of narrow-band single LED light sources, or a plurality of lasers with good monochromaticity; the structured light generating and modulating module comprises a beam splitter, a grating, a digital micro-mirror device or a spatial light modulator which are cascaded; the polarization control module is a polarization rotator consisting of a variable phase retarder and a quarter-wave plate or a half-wave plate fixed on a rotating device and is used for rotating the linear polarization direction of the light source to obtain high-contrast structured light stripes in a light beam overlapping area.

2. The high-speed multicolor multi-modal structured light illuminated super-resolution microscopy imaging system according to claim 1, wherein: the structured light generation and modulation module is configured to generate at least two beams of coherent or semi-coherent light that, when intersected within a sample, generate periodic structured light within an overlap region for illuminating the sample.

3. The high-speed multicolor multi-modal structured light illuminated super-resolution microscopy imaging system according to claim 1, wherein: when a monochromatic laser is adopted, the high-speed gating switching module is an acousto-optic tunable filter or an electro-optic modulator and is used for selecting one or more lasers with specific wavelengths to be coupled into a subsequent optical path;

4. The high-speed multicolor multi-modal structured light illuminated super-resolution microscopy imaging system according to claim 1, wherein: the spatial filtering module comprises a high-speed vibration motor and a filtering template; the filtering template is installed on the high-speed vibration motor and used for filtering redundant stray light of the structural light stripe in a specific direction.

5. The high-speed multicolor multi-modal structured light illuminated super-resolution microscopy imaging system according to claim 1, wherein: the fluorescence module comprises a cylindrical mirror, a dichroic sheet and an optical filter; the detection module comprises a scientific research camera for detecting signal light and a filter turntable for selecting a proper filter for a specific signal light wavelength range to remove exciting light.

6. The high-speed multicolor multi-modal structured light illuminated super-resolution microscopy imaging system according to claim 1, wherein: the detection module at least comprises two paths, wherein when one path is collected, the other path prepares a corresponding optical filter in advance aiming at the spectral range of the next signal light, and the non-stop collection is realized through alternate operation.

7. The imaging method of the high-speed multi-color multi-modal structured light illuminated super-resolution microscopy imaging system as recited in any one of claims 1 to 6, wherein: under the light illumination mode of the total internal reflection, grazing incidence or falling incidence structure formed by double light beam incidence, the method comprises the following steps:

8. The imaging method of the high-speed multicolor multi-modal structured light illuminated super-resolution microscopy imaging system as claimed in any one of claims 1 to 6, wherein in the three-beam incident forming epi-structured light illumination mode, the method comprises the following steps: