Lattice structured light illumination microscope based on three-beam interference and implementation method

Abstract

The present invention discloses a lattice structured light illumination microscope based on three-beam interference and its implementation method. The present invention controls the hexagonal lattice pattern formed by the beam modulation system so that the diffracted light after phase modulation passes through a spatial mask, retaining only three beams of +1 or three beams of ‑1 order diffracted light. The fast axis direction of each area of ​​the polarization modulator is different, and the polarization state of each diffracted beam is independently adjusted. The polarization state of each diffracted light beam is radial polarization, and the interference pattern irradiated on the sample is a hexagonal lattice pattern, which enhances the amplitude of high-frequency information. An adaptive compensation filter is used for the spectrum of the SIM image to effectively reduce artifacts caused by severe background in the SIM reconstructed image. While ensuring the expansion of isotropic resolution, the redundancy of the collected information is reduced, the phototoxicity is reduced, and the imaging time of the sample is extended.

Description

Technical Field

[0001] The present invention relates to a super-resolution fluorescence microscopy technology, and in particular to a lattice structure light illumination microscope based on three-beam interference and an implementation method thereof. Background Art

[0002] Structured illumination super-resolution microscopy (SIM) has become a leading approach for observing living cells and the interactions within their internal organelles. This is primarily due to its lower excitation light requirements compared to other super-resolution fluorescence microscopy methods, such as stimulated emission depletion (STED) and stochastic optical reconstruction super-resolution microscopy (STORM), and its compatibility with both traditional fluorescent dyes and fluorescent proteins. The widespread application of SIM has provided new mechanistic insights into numerous biological processes.

[0003] The main principle of structured light super-resolution microscopy is that when structured light is irradiated on a sample, the subtle structure in the sample interacts with the fringe pattern of the illumination, generating low-frequency moiré fringes. These moiré fringes contain modulations of high-frequency information in the sample, enabling the capture of higher-resolution details during imaging than is possible with traditional microscopy.

[0004] Traditional SIM uses stripe structured light for illumination, but a single illumination shot can only achieve resolution expansion in one direction. To achieve isotropic resolution, the stripes must be rotated in three directions, shifting the phase by three steps in each direction. This results in repeated widefield image acquisition in each direction, creating information redundancy, increasing photobleaching, and shortening continuous imaging time.

[0005] Compared to stripe structured light illumination, which is modulated based on one-dimensional illumination, lattice structured light illumination is modulated based on two-dimensional illumination. Common lattice illumination methods include orthogonal lattice illumination and hexagonal lattice illumination. Orthogonal lattice illumination requires five original images for reconstruction, but suffers from anisotropic resolution, while hexagonal lattice illumination requires seven original images for reconstruction, but maintains isotropic resolution.

[0006] The advantage of two-dimensional modulation based on lattice illumination is that it can achieve isotropic resolution expansion with a single illumination shot, avoiding the information redundancy associated with reconstructing and acquiring widefield images. However, its disadvantage is the reduction in the amplitude of high-frequency information. This limits the potential of lattice illumination-based super-resolution fluorescence microscopy.

[0007] Similar to traditional fringe-modulated two-dimensional structured illumination microscopy (2D-SIM), triple-beam interferometric SIM (3I-SIM) uses modulated illumination to excite fluorophores in the sample plane:

[0008]

[0009]

[0010] Here I n (r) represents the distribution of the illumination pattern generated by interference after the nth phase shift, which requires 7 shifts in total. I0 represents the unit intensity of the incident light, k j Represents the frequency of the cosine function in the jth direction. Represents the distance of the nth phase shift in the jth direction, m j represents the amplitude of the cosine function, and r represents the spatial coordinate.

[0011] After being excited by the structured illumination light, the detected signal should be the convolution of the system point spread function (PSF) and the modulation signal:

[0012]

[0013] D n (r) is the signal of the sample collected by the camera after being modulated by structured light illumination and point spread function, s(r) represents the signal distribution of the object, I n (r) represents the distribution of the nth illumination image, and h(r) is the point spread function of the system.

[0014] Using the Fourier view, the 3I-SIM super-resolution image is reconstructed by transforming equations (1-3) into the Fourier domain:

[0015]

[0016] D n (k) is the frequency domain distribution of the object signal distribution signal, I n (k) is the frequency domain distribution of the illumination pattern, H(k) is the optical transfer function (OTF) of the system, and k is the frequency domain coordinate.

[0017] When using stripe structured light illumination, reconstruction requires nine stripe images in three directions and three phases, for a total of nine. While rolling reconstruction allows for reuse of the original images, for stripe structured light illumination, rolling reconstruction is required group by group because phases in the same direction must be aligned. Using single-frame rolling reconstruction causes the phase images of one group to search for the phase images of the previous group in the same direction. This results in original images in the same direction not being captured at adjacent times, leading to motion artifacts.

[0018] When using lattice structured light illumination, only seven phase images in one direction are required for reconstruction, for a total of seven images. Rolling reconstruction allows the original images to be reused. For 3I-SIM, single-frame rolling reconstruction is possible. This is because the acquired phase images do not need to be aligned in direction.

[0019] Different from the commonly used one-dimensional linear cosine illumination pattern, the function used to construct the hexagonal illumination pattern contains three cosine terms. Each cosine term contains two unknown frequency components S(k±k j )(i=1,2,3) and mutual zero-frequency components S, as well as seven unknowns.

[0020] Therefore, it is necessary to collect seven images and construct a set of seven-variable linear equations:

[0021]

[0022] To reconstruct the super-resolution image, a translation matrix M is constructed; then, the seven frequency components are calculated by the following formula:

[0023]

[0024] After obtaining the separated spectra, the spectra can be shifted and superimposed to obtain a super-resolution image.

[0025] The directly superimposed spectrum is abnormal, so Wiener deconvolution and OTF attenuation are often used to reduce the artifacts caused by spectral anomalies. Although Wiener deconvolution and OTF attenuation can solve background-related artifacts, in severe backgrounds, the final optimized SIM image still tends to have some residual background-related hexagonal artifacts. The mechanism is that the Wiener deconvolution will increase the OTF attenuation part, so that the resulting image still contains a lot of out-of-focus information. Although increasing the Wiener parameter w can reduce the degree of increased OTF attenuation to a certain extent, it will still suppress the entire high-frequency part, resulting in a lower signal-to-noise ratio. Summary of the Invention

[0026] In response to the problems existing in the above-mentioned prior art, the present invention proposes a lattice structured light illumination microscope based on three-beam interference and its implementation method, which adopts an adaptive compensation filter instead of OTF attenuation to obtain a super-resolution image with background artifacts removed, effectively eliminating artifacts caused by severe background in SIM reconstructed images.

[0027] One object of the present invention is to provide a lattice structured light illumination microscope based on three-beam interference.

[0028] The lattice structure light illumination microscope based on three-beam interference of the present invention comprises: a laser, a gating device, a beam modulation system, a Fourier transform system, a spatial filtering device, a polarization modulator, a dichroic mirror, an objective lens, a tube lens and a camera; wherein the laser outputs laser light of multiple wavelengths, and the wavelength of the laser light is selected and the intensity of the laser light is controlled by the gating device; the linearly polarized light beam is passed through the beam modulation system to perform phase modulation on the incident parallel light, so that the incident parallel light is diffracted, and by controlling the hexagonal lattice pattern formed in the beam modulation system, the diffracted light after phase modulation has at least three beams of +1 order diffracted light or three beams of -1 order diffracted light, and the diffracted light emitted from the beam modulation system is sent to the Fourier transform system; The Fourier transform system focuses the diffracted light onto the Fourier plane of the Fourier transform system; the unnecessary zero-order and redundant-order diffracted light is filtered out through a spatial filtering device, and only three beams of +1-order diffracted light or three beams of -1-order diffracted light are retained to the polarization modulator; the polarization modulator is divided into at least three uniform areas along the angle, and the polarization modulation direction of each area is different. Each beam of +1-order diffracted light or -1-order diffracted light is incident on the corresponding area respectively, and the polarization direction of each beam of diffracted light is accurately controlled by the polarization modulator without losing the laser power. The polarization state of each beam of diffracted light is adjusted according to the incident angle of the interference light irradiated on the sample surface, so that the polarization state of each beam of diffracted light passing through the polarization modulator is The three diffracted beams of radial polarization pass through the dichroic mirror to the objective lens to ensure that the polarization of the incident light does not change; the objective lens converts the focused diffracted light into parallel light, and the radially polarized incident light is irradiated on the sample at an incident angle greater than 54.7° to cause interference; the fluorescence emitted by the sample is emitted through the same objective lens, and is focused onto the target surface of the camera through the dichroic mirror and the tube lens, and is transmitted to the computer to obtain the SIM original image. The diffracted light at a large angle is incident with radial polarization, so that the signal-to-noise ratio of the collected SIM original image is the best; by changing the hexagonal lattice pattern on the beam modulation system, the interference pattern on the sample is phase shifted, and each phase shift obtains a SIM original image, and a total of seven images are obtained. SIM original images are reconstructed as a group; seven SIM original images are preprocessed, parameter estimated, spectral separated, spectral shifted and spectral superimposed to obtain the spectrum of the SIM image; an adaptive compensation filter is used on the spectrum of the SIM image to notch the spectral region around the offset frequency point, and the amplitude at the offset frequency point is compared with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal double super-resolution image. The amplitude at the offset frequency point is adjusted to make it consistent with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal double super-resolution image, effectively removing background-related artifacts and obtaining a super-resolution image with background artifacts removed.

[0029] Furthermore, the present invention also includes a beam expansion and shaping device. After passing through the gating device, the laser beam is expanded and mode-shaped by the beam expansion and shaping device, and light of other modes except the fundamental mode is filtered out to ensure the uniformity of the laser, and the divergent light is converted into parallel light. The polarization state of the beam after beam expansion and shaping is linear polarization; the beam expansion and shaping device adopts a first and a second lens and a shaping element. The shaping element adopts a single-mode polarization-maintaining optical fiber, a pinhole or a light vibration device to eliminate spatial coherence and suppress speckle on the imaging surface; the first lens focuses the laser into the shaping element, and the shaping element can filter out light of other modes except the fundamental mode, so as to ensure the mode of the illumination light and improve the visibility of the interference fringes; the polarization state of the light beam emitted by the shaping element is linear polarization; after the laser is output through the shaping element, the second lens converts the divergent light into parallel light.

[0030] The gating device adopts an acousto-optic tunable filter.

[0031] The beam modulation system uses a polarization beam splitter, a half-wave plate and a liquid crystal spatial light modulator, or a digital microlens array, or a diffraction optical element, or a triangular or hexagonal grating; using a polarization beam splitter, a half-wave plate and a liquid crystal spatial light modulator: a straight polarized light beam is reflected by the polarization beam splitter and passes through the half-wave plate to the liquid crystal spatial light modulator; the half-wave plate and the polarization beam splitter form a phase grating to phase modulate the incident parallel light, causing the incident parallel light to diffract. By controlling the hexagonal lattice pattern loaded on the liquid crystal spatial light modulator, the phase-modulated diffracted light has at least three beams of +1-order diffracted light or three beams of -1-order diffracted light. The diffracted light emitted from the liquid crystal spatial light modulator passes through the half-wave plate, and its polarization state becomes horizontal polarization, and is transmitted through the polarization beam splitter; using a digital micromirror array: the incident light is controlled by the digital micromirror array, and the diffraction contains different diffraction orders. The digital micromirror array does not change the polarization state of the incident light. Using a diffractive optical element: A diffractive optical element is a passive device that achieves the desired diffraction order by changing the distribution of the optical material within it. The +1st-order diffracted light or -1st-order diffracted light is arranged in a triangular pattern, forming an equilateral triangle distribution.

[0032] The Fourier transform system uses a third lens.

[0033] The polarization direction of the diffraction pattern arranged in a triangle is distributed radially. The polarization modulator uses a multi-section half-wave plate or a multi-section polarizer.

[0034] The spatial filtering device adopts a mask plate.

[0035] It also includes a conjugate system, through which three beams of radially polarized diffracted light are conjugated to the back focal plane of the objective lens; the conjugate system uses a pair of coupling lenses or a pair of concave reflecting mirrors, and the pair of coupling lenses includes a fourth lens and a fifth lens.

[0036] The laser uses multiple single-mode lasers to form multi-color lasers through optical fiber or free space coupling.

[0037] The adaptive compensation filter adopts the filter form, the adaptive compensation filter G adaptive (k) The mathematical description is as follows:

[0038]

[0039] Among them, S SIM is the spectrum of the SIM image, H is the optical transfer function of the widefield image, k is the spatial coordinate, k i is the center position of the notch, c is the penalty coefficient, and attWidth is the width of the filter.

[0040] When the incident angle of the interference light irradiating the sample surface is less than 54.7°, the high-frequency information amplitude corresponding to circular polarization and the signal-to-noise ratio of the collected image are optimal. When the incident angle is equal to 54.7°, the high-frequency information amplitude corresponding to circular polarization, angular polarization, and radial polarization and the signal-to-noise ratio of the collected image are the same. When the incident angle is greater than 54.7°, the high-frequency information amplitude corresponding to radial polarization and the signal-to-noise ratio of the collected image are optimal. Under the high spread spectrum conditions of the super-resolution imaging of the present invention, radial polarization is the optimal choice for large-angle interference light incidence. Large angle refers to an incident angle greater than 54.7°.

[0041] Another object of the present invention is to provide a method for realizing a lattice structured light illumination microscope based on three-beam interference.

[0042] The method for realizing a lattice structured light illumination microscope based on three-beam interference of the present invention comprises the following steps:

[0043] 1) The laser outputs multiple wavelengths of laser light, and the wavelength of the laser light is selected and the intensity of the laser light is controlled by the gating device;

[0044] 2) The linearly polarized light beam passes through a beam modulation system to phase modulate the incident parallel light, causing the incident parallel light to diffract. By controlling the hexagonal lattice pattern formed in the beam modulation system, the diffracted light after phase modulation has at least three +1-order diffracted lights or three -1-order diffracted lights. The diffracted light emitted from the beam modulation system is sent to a Fourier transform system.

[0045] 3) The Fourier transform system focuses the diffracted light onto the Fourier plane of the Fourier transform system;

[0046] 4) Using a spatial filter device, the unnecessary zero-order and extra-order diffraction light is filtered out, and only three beams of +1-order diffraction light or three beams of -1-order diffraction light are retained to the polarization modulator;

[0047] 5) The polarization modulator is divided into at least three uniform regions along the radial direction, and the polarization modulation direction of each region is different.

[0048] The +1-order diffracted light or -1-order diffracted light is incident on the corresponding area respectively. The polarization direction of each diffracted light beam is precisely controlled by the polarization modulator without losing laser power. The polarization state of each diffracted light beam is adjusted according to the incident angle of the interference light irradiated on the sample surface, so that the polarization state of each diffracted light beam passing through the polarization modulator is radial polarization.

[0049] 6) The three radially polarized diffracted beams pass through a dichroic mirror to the objective lens to ensure that the polarization of the incident light does not change;

[0050] 7) The objective lens converts the focused diffracted light into parallel light. The radially polarized incident light shines on the sample at an incident angle greater than 54.7°, causing interference. The interference pattern is a hexagonal lattice pattern.

[0051] 8) Fluorescence emitted by the sample is emitted through the same objective lens, passed through a dichroic mirror and a tube lens, focused onto the target surface of the camera, and transmitted to the computer to obtain a SIM raw image. The diffracted light at a large angle is incident with radial polarization, so that the signal-to-noise ratio of the collected SIM raw image is optimized;

[0052] 9) By changing the hexagonal lattice pattern on the beam modulation system, the interference pattern on the sample is phase shifted. Each phase shift generates a SIM raw image, and a total of seven SIM raw images are obtained as a group for reconstruction;

[0053] 10) Preprocessing, parameter estimation, spectrum separation, spectrum shifting, and spectrum superposition are performed on the seven SIM original images to obtain the spectrum of the SIM image;

[0054] 11) An adaptive compensation filter is applied to the spectrum of the SIM image to notch the spectrum region around the offset frequency point. The amplitude at the offset frequency point is compared with a scaled 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal double super-resolution image. The amplitude at the offset frequency point is adjusted so that the amplitude at the offset frequency point is consistent with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal double super-resolution image, thereby effectively removing background-related artifacts and obtaining a super-resolution image with background artifacts removed.

[0055] In step 2), the beam modulation system uses a polarization beam splitter, a half-wave plate, and a liquid crystal spatial light modulator, or a digital microlens array, or a diffractive optical element, or a triangular or hexagonal grating. The +1-order diffracted light or the -1-order diffracted light is arranged in a triangular shape, forming an equilateral triangle distribution.

[0056] In step 5), the polarization direction of the diffraction pattern arranged in a triangle is distributed radially.

[0057] In step 11), the adaptive compensation filter adopts the form of Gaussian filter, and the adaptive compensation filter G adaptive (k) The mathematical description is as follows:

[0058]

[0059] Among them, S SIM is the spectrum of the SIM image, H is the optical transfer function of the widefield image, k is the spatial coordinate, k i is the center position of the notch, c is the penalty coefficient, and attWidth is the width of the filter.

[0060] Advantages of the present invention:

[0061] The present invention controls the hexagonal lattice pattern formed by the beam modulation system so that the phase-modulated diffracted light passes through the spatial mask, retaining only three beams of +1 or three beams of -1 order diffracted light; the polarization modulation direction of each area of ​​the polarization modulator is different, and the polarization direction of each beam of diffracted light is precisely controlled. The polarization state of each beam of diffracted light is radial polarization, and the interference pattern irradiated on the sample is a hexagonal lattice pattern; an adaptive compensation filter is used for the spectrum of the SIM image to effectively remove artifacts caused by severe background in the SIM reconstructed image; the hexagonal lattice illumination principle is adopted to reduce the redundancy of the collected information and extend the imaging time while ensuring the expansion of isotropic resolution; the three-partition half-wave plate independently adjusts the polarization state of each diffracted light beam, thereby enhancing the amplitude of high-frequency information; through theoretical analysis, the present invention points out that under different expansion frequencies of structured light illumination, the corresponding optimal polarization state is different, which expands the applicability of lattice illumination microscopes. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is a schematic diagram of an embodiment of a lattice structured light illumination microscope based on three-beam interference of the present invention;

[0063] Figure 2 Schematic diagram of the polarization state in the optical path of an embodiment of a lattice structured light illumination microscope based on three-beam interference of the present invention;

[0064] Figure 3 A schematic diagram of a three-partition half-wave plate of an embodiment of a lattice structured light illumination microscope based on three-beam interference of the present invention;

[0065] Figure 4 Interference pattern diagrams on a sample surface corresponding to different polarization states obtained by an embodiment of a lattice structured light illumination microscope based on three-beam interference of the present invention;

[0066] Figure 5 The figure is a signal-to-noise ratio curve diagram of an embodiment of the lattice structured light illumination microscope based on three-beam interference of the present invention. DETAILED DESCRIPTION

[0067] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

[0068] like Figure 1 As shown, the lattice structured light illumination microscope based on three-beam interference of this embodiment includes: a laser, an acousto-optic tunable filter, a beam expansion and shaping device, a polarization beam splitter, a half-wave plate, a liquid crystal spatial light modulator, a Fourier transform system, a mask, a three-partition half-wave plate, a conjugate system, a polarization-maintaining dichroic mirror, an objective lens, a tube lens and a camera; wherein, the laser uses multiple monochromatic lasers to output monochromatic lasers and then couple them into multi-color lasers, outputting lasers of multiple wavelengths, and the acousto-optic tunable filter is used to select the wavelength of the laser and control the intensity of the laser; the beam expansion and shaping device uses the first and second lenses and a single-mode polarization-maintaining fiber; the first lens focuses the laser into the single-mode polarization-maintaining fiber, and the single-mode polarization-maintaining fiber can filter out light of other modes except the fundamental mode, so as to ensure the uniformity of the illumination light, and the polarization state of the light beam emitted by the single-mode polarization-maintaining fiber is vertical polarization. After the laser is output through the single-mode polarization-maintaining fiber, the second lens converts the divergent light into parallel light, and the polarization state of the light beam after beam expansion and shaping is The vertical polarization state is vertical; the vertically polarized light beam is reflected by the polarization beam splitter and passes through the half-wave plate to the liquid crystal spatial light modulator; the half-wave plate and the polarization beam splitter form a phase grating to phase modulate the incident parallel light, causing the incident parallel light to diffract. By controlling the hexagonal lattice pattern loaded on the liquid crystal spatial light modulator, the diffracted light after phase modulation has three +1 order diffracted lights, three -1 order diffracted lights, and zero order and other order diffracted lights. The incident light passes through the half-wave plate, and its polarization state becomes horizontal polarization, and is transmitted through the polarization beam splitter; the Fourier transform system uses a third lens, which focuses the diffracted light onto the Fourier plane of the third lens; the unnecessary zero-order and redundant-order diffracted light is filtered out through a spatial mask, and only three beams of +1-order diffracted light or three beams of -1-order diffracted light are retained to the three-partition half-wave plate. The arrangement of the +1-order diffracted light or -1-order diffracted light is triangular, forming an equilateral triangle distribution; the polarization state in the optical path is as follows Figure 2 As shown; Figure 3As shown in the figure, the three-part half-wave plate is divided into three uniform areas along the radial direction, and the polarization modulation direction of each area is different. Each beam of +1 order diffraction light or -1 order diffraction light is incident on the corresponding area respectively. The polarization direction of each beam of diffraction light is accurately controlled by the three-part half-wave plate without losing laser power, so that the polarization state of each beam of diffraction light passing through the three-part half-wave plate is radial polarization; the three beams of radially polarized diffraction light are conjugated to the back focal plane of the objective lens through a pair of coupling lenses; the conjugate system adopts a pair of coupling lenses, including the fourth and fifth lenses, and the diffraction light focused by the pair of coupling lenses passes through the polarization-maintaining dichroic mirror to the objective lens to ensure that the polarization of the incident light does not change; the objective lens converts the focused diffraction light into parallel light, which is irradiated on the sample to cause interference, and the interference pattern is a hexagonal lattice pattern; the fluorescence emitted by the sample is emitted through the same objective lens, and is focused onto the target surface of the camera through the polarization-maintaining dichroic mirror and the tube lens, and is transmitted The SIM original image is input to a computer; the interference pattern on the sample is phase-shifted by transforming the hexagonal lattice pattern on the liquid crystal spatial light modulator, and a SIM original image is obtained each time the phase shift occurs. A total of seven SIM original images are reconstructed as a group; the seven SIM original images are preprocessed, parameter estimated, spectral separated, spectral shifted and spectral superimposed to obtain the spectrum of the SIM image; an adaptive compensation filter is used for the spectrum of the SIM image to notch the spectrum area around the offset frequency point, and the amplitude at the offset frequency point is compared with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal twice super-resolution image, and the size of the amplitude at the offset frequency point is adjusted so that the amplitude at the offset frequency point is consistent with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal twice super-resolution image, thereby effectively removing background-related artifacts and obtaining a super-resolution image with background artifacts removed.

[0069] In single-shot illumination, lattice illumination modulates high-frequency information with a weaker amplitude than stripe illumination. Consequently, in low-signal-to-noise ratio images, illumination parameter estimation is prone to bias and error, limiting the widespread application of lattice illumination strategies. This method, by adjusting the polarization state of diffracted light to radial polarization, increases the high-frequency information modulation amplitude by approximately 50% compared to the original angular polarization under high-spread spectrum conditions.

[0070] The influence of different polarization states on the interference pattern of the sample surface at different incident angles:

[0071] (1) Angular polarization

[0072] When the three polarized beams on the sample surface are angularly polarized, the intensity distribution of the interference pattern on the sample surface is as follows:

[0073]

[0074] Iap represents the intensity distribution of the interference pattern under angular polarization, 3 is the intensity of the DC component, the e index represents the high-frequency component, k represents the frequency of the AC component, and x and y represent the coordinates on the plane.

[0075] Its high frequency modulation amplitude is calculated as:

[0076]

[0077] (2) Circular polarization

[0078] When the three polarized lights on the sample surface are circularly polarized, the intensity distribution of the interference pattern on the sample surface is as follows:

[0079]

[0080] I cp represents the intensity distribution of the interference pattern under circular polarization, 3 is the intensity of the DC component, the e index represents the high-frequency component, k represents the frequency of the AC component, x and y represent the coordinates on the plane, and θ is the angle between the incident light and the normal of the sample plane.

[0081] The high frequency modulation amplitude is calculated as:

[0082]

[0083] Through numerical calculation, the results can be obtained as follows:

[0084]

[0085] Further numerical calculations verify that when θ < 54.7, m cp >1 / 6=m ap , and when θ>54.7, m cp <1 / 6=m ap

[0086] (3) Radial polarization

[0087] When the three polarized lights on the sample surface are radially polarized, the intensity distribution of the interference pattern on the sample surface is as follows:

[0088]

[0089] I rp represents the intensity distribution of the interference pattern under circular polarization, 3 is the intensity of the DC component, the e index represents the high-frequency component, k represents the frequency of the AC component, x and y represent the coordinates on the plane, and θ is the angle between the incident light and the normal of the sample plane.

[0090] The high frequency modulation amplitude is calculated as:

[0091]

[0092] Through numerical calculation, we can further obtain that when θ>54.7, m rp >1 / 6=m ap , and when m cp <1 / 6=m ap .

[0093] Figure 4 The interference patterns on the sample surface corresponding to different polarization states are shown. The interference pattern of radial polarization is different from the other two because the phase is reversed.

[0094] Combining the above numerical calculations, we can get the corresponding intensity of the high-frequency information amplitude at different angles and different polarization states, which also represents the signal-to-noise ratio of the image that can be obtained in each case, such as Figure 5 shown.

[0095] from Figure 5 The following conclusions can be drawn: when the incident angle of the interference light irradiating the sample surface is less than 54.7°, the high-frequency information amplitude corresponding to circular polarization and the signal-to-noise ratio of the acquired image are optimal; when the incident angle is equal to 54.7°, the high-frequency information amplitude and the signal-to-noise ratio of the acquired image are the same in all three cases; when the incident angle is greater than 54.7°, the high-frequency information amplitude corresponding to radial polarization and the signal-to-noise ratio of the acquired image are optimal. Therefore, radial polarization is the optimal choice under high spread spectrum conditions.

[0096] Finally, it should be noted that the purpose of disclosing the embodiments is to facilitate a further understanding of the present invention. However, those skilled in the art will appreciate that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the contents disclosed in the embodiments; the scope of protection claimed by the present invention shall be determined by the scope defined in the claims.

Claims

1. A lattice structured light illumination microscope based on three-beam interference, characterized in that: The lattice structure light illumination microscope includes: a laser, a gating device, a beam modulation system, a Fourier transform system, a spatial filtering device, a polarization modulator, a dichroic mirror, an objective lens, a tube lens and a camera; wherein the laser outputs lasers of multiple wavelengths, and the wavelength of the laser is selected and the intensity of the laser is controlled by the gating device; the linearly polarized light beam is passed through the beam modulation system to perform phase modulation on the incident parallel light, so that the incident parallel light is diffracted, and the hexagonal lattice pattern formed in the beam modulation system is controlled so that the diffracted light after phase modulation has at least three beams of +1 order diffracted light or three beams of -1 order diffracted light, and the diffracted light emitted from the beam modulation system is sent to the Fourier transform system; the Fourier transform system converts the diffracted light Focus on the Fourier plane of the Fourier transform system; filter out the unnecessary zero-order and redundant-order diffraction light through the spatial filtering device, and only retain three beams of +1-order diffraction light or three beams of -1-order diffraction light to the polarization modulator; the polarization modulator is divided into at least three uniform areas along the angle, and the polarization modulation direction of each area is different. Each beam of +1-order diffraction light or -1-order diffraction light is incident on the corresponding area respectively, and the polarization direction of each beam of diffraction light is accurately controlled by the polarization modulator without losing the laser power. The polarization state of each beam of diffraction light is adjusted according to the incident angle of the interference light irradiated on the sample surface, so that the polarization state of each beam of diffraction light passing through the polarization modulator is radial polarization; three beams The radially polarized diffracted light passes through the dichroic mirror to the objective lens to ensure that the polarization of the incident light does not change; the objective lens converts the focused diffracted light into parallel light, and the radially polarized incident light is irradiated on the sample at an incident angle greater than 54.7° to cause interference; the fluorescence emitted by the sample is emitted through the same objective lens, and is focused onto the target surface of the camera through the dichroic mirror and the tube lens, and is transmitted to the computer to obtain the structured light illumination super-resolution microscope SIM original image. The diffracted light at a large angle is incident with radial polarization, so that the signal-to-noise ratio of the collected SIM original image is the best; by changing the hexagonal lattice pattern on the beam modulation system, the interference pattern on the sample is phase shifted, and each phase shift obtains a SIM original image, and a total of seven The seven SIM original images are reconstructed as a group; the seven SIM original images are preprocessed, parameter estimated, spectral separated, spectral shifted and spectral superimposed to obtain the spectrum of the SIM image; the spectrum of the SIM image is notched by an adaptive compensation filter around the offset frequency point, the amplitude at the offset frequency point is compared with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal twice super-resolution image, and the amplitude at the offset frequency point is adjusted to make it consistent with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal twice super-resolution image, effectively removing background-related artifacts and obtaining a super-resolution image with background artifacts removed.

2. The lattice structured light illumination microscope according to claim 1, characterized in that: It also includes a beam expansion and shaping device. After the laser passes through the gating device, the laser beam is expanded and mode-shaped by the beam expansion and shaping device, and light of other modes except the fundamental mode is filtered out to ensure the uniformity of the laser, and the divergent light is converted into parallel light. The polarization state of the beam after beam expansion and shaping is linear polarization; the beam expansion and shaping device adopts the first and second lenses and the shaping element, and the shaping element adopts a single-mode polarization-maintaining optical fiber, a pinhole or a light vibration device.

3. The lattice structured light illumination microscope according to claim 1, wherein: The invention comprises a conjugate system, wherein three beams of radially polarized diffracted light are conjugated to the back focal plane of the objective lens through the conjugate system; the conjugate system adopts a pair of coupling lenses or a pair of concave reflecting mirrors.

4. The lattice structured light illumination microscope according to claim 1, wherein: The light beam modulation system adopts a polarization beam splitter, a half-wave plate and a liquid crystal spatial light modulator, or adopts a digital micro lens array, or adopts a diffraction optical element.

5. The lattice structured light illumination microscope according to claim 1, wherein: The laser uses multiple single-mode lasers to form multi-color lasers through optical fiber or free space coupling.

6. The lattice structured light illumination microscope according to claim 1, wherein: The adaptive compensation filter adopts the form of Gaussian filter, and the adaptive compensation filter G adaptive (k) The mathematical description is as follows: Among them, S SIM is the spectrum of the SIM image, H is the optical transfer function of the widefield image, k is the spatial coordinate, k i is the position of the center of the notch, c is the penalty coefficient, and attWidth is the width of the filter.

7. A method for realizing a lattice structured light illumination microscope based on three-beam interference as claimed in claim 1, characterized in that: The implementation method comprises the following steps: 1) The laser outputs multiple wavelengths of laser light, and the wavelength of the laser light is selected and the intensity of the laser light is controlled by the gating device; 2) The linearly polarized light beam passes through a beam modulation system to phase modulate the incident parallel light, causing the incident parallel light to diffract. By controlling the hexagonal lattice pattern formed in the beam modulation system, the diffracted light after phase modulation has at least three +1-order diffracted lights or three -1-order diffracted lights. The diffracted light emitted from the beam modulation system is sent to a Fourier transform system. 3) The Fourier transform system focuses the diffracted light onto the Fourier plane of the Fourier transform system; 4) Using a spatial filter device, the unnecessary zero-order and extra-order diffraction light is filtered out, and only three beams of +1-order diffraction light or three beams of -1-order diffraction light are retained to the polarization modulator; 5) The polarization modulator is divided into at least three uniform regions along the radial direction, each region having a different polarization modulation direction. Each beam of +1-order diffracted light or -1-order diffracted light is incident on the corresponding region. The polarization direction of each diffracted light beam is precisely controlled by the polarization modulator without losing laser power. The polarization state of each diffracted light beam is adjusted according to the incident angle of the interference light irradiated on the sample surface, so that the polarization state of each diffracted light beam passing through the polarization modulator is radial polarization. 6) The three radially polarized diffracted beams pass through a dichroic mirror to the objective lens to ensure that the polarization of the incident light does not change; 7) The objective lens converts the focused diffracted light into parallel light. The radially polarized incident light shines on the sample at an incident angle greater than 54.7°, causing interference. The interference pattern is a hexagonal lattice pattern. 8) The fluorescence emitted by the sample is emitted through the same objective lens, passed through the dichroic mirror and the tube lens, and focused onto the target surface of the camera. The SIM original image is transmitted to the computer, and the diffracted light with a large angle is incident with radial polarization, so that the signal-to-noise ratio of the collected SIM original image is optimal; 9) By changing the hexagonal lattice pattern on the beam modulation system, the interference pattern on the sample is phase shifted. Each phase shift generates a SIM raw image, and a total of seven SIM raw images are obtained as a group for reconstruction; 10) Preprocessing, parameter estimation, spectrum separation, spectrum shifting, and spectrum superposition are performed on the seven SIM original images to obtain the spectrum of the SIM image; 11) Adaptive compensation filter is used for the spectrum of SIM image to notch the spectrum area around the offset frequency point. The amplitude at the offset frequency point is compared with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal double super-resolution image, and the amplitude at the offset frequency point is adjusted so that the amplitude at the offset frequency point is consistent with 1 / 2 of the optical transfer function (OTF) of the wide-field image corresponding to the ideal double super-resolution image, effectively removing background-related artifacts and obtaining a super-resolution image with background artifacts removed.

8. The implementation method according to claim 7, characterized in that: In step 2), the beam modulation system uses a polarization beam splitter, a half-wave plate and a liquid crystal spatial light modulator, or a digital microlens array, or a diffractive optical element, or a triangularly arranged or hexagonally arranged grating.

9. The implementation method according to claim 7, wherein: In step 4), the +1st order diffraction light or the -1st order diffraction light is arranged in a triangular shape, forming an equilateral triangle distribution.

10. The implementation method according to claim 7, wherein: In step 11), the adaptive compensation filter adopts the form of Gaussian filter, and the adaptive compensation filter G adaptive (k) The mathematical description is as follows: Among them, S SIM is the spectrum of the SIM image, H is the optical transfer function of the widefield image, k is the spatial coordinate, k i is the center position of the notch, c is the penalty coefficient, and attWidth is the width of the filter.

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