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

Abstract

The invention discloses a lattice structure light illumination microscope based on three-beam interference and an implementation method. By controlling the hexagonal lattice pattern formed by the light beam modulation system, the diffracted light after phase modulation passes through the space mask, and only three + 1-level or three-1-level diffracted light is reserved; the fast axis directions of all areas of the polarization modulator are different, the polarization state of each diffracted light beam is independently adjusted, the polarization state of each diffracted light beam is radial polarization, a pattern irradiated on a sample to generate interference is a hexagonal lattice pattern, and the amplitude of high-frequency information is enhanced; a self-adaptive compensation filter is adopted for the frequency spectrum of the SIM image, so that artifacts caused by a serious background in the SIM reconstructed image are effectively reduced; while isotropic resolution expansion is ensured, redundancy of acquired information is reduced, and phototoxicity is reduced, so that imaging time of a sample is prolonged.

Description

Technical Field

[0001] The 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 a realization method thereof. Background Art

[0002] Structured illumination super-resolution microscopy (SIM) has become the main means of observing living cells and the interactions between their internal organelles. This is mainly due to the fact that compared with other super-resolution fluorescence microscopy, such as stimulated emission depletion super-resolution microscopy (STED) and stochastic optical reconstruction super-resolution microscopy (STORM), SIM has smaller requirements for excitation light and is compatible with traditional fluorescent dyes and fluorescent proteins. The wide application of SIM has provided new mechanistic insights into many biological processes.

[0003] The main principle of structured light illumination super-resolution microscopy: When structured light is irradiated on a sample, the fine structure in the sample interacts with the stripe pattern of the light, generating low-frequency moiré fringes. These moiré fringes contain the modulation of high-frequency information in the sample, thus capturing higher resolution details than traditional microscopy during imaging.

[0004] Traditional SIM uses stripe structured light for illumination, and a single illumination can only achieve resolution expansion in one direction. To achieve isotropic resolution, the stripes need to be rotated in three directions, with three phases shifted in each direction. This results in repeated acquisition of wide-field images in each direction, resulting in information redundancy, which increases photobleaching and shortens the time of continuous imaging.

[0005] Compared with stripe structured light illumination, which is modulated based on one-dimensional illumination, lattice structured light illumination is modulated based on two-dimensional illumination. Commonly used lattice illuminations include orthogonal lattice illumination and hexagonal lattice illumination. Orthogonal lattice illumination requires 5 original images for reconstruction, but the resolution is anisotropic, while hexagonal lattice illumination requires 7 original images for reconstruction, but the resolution is isotropic.

[0006] The advantage of two-dimensional modulation based on lattice illumination is that it can achieve isotropic resolution expansion under single illumination and avoid the information redundancy caused by reconstructing and collecting wide-field images. However, its disadvantage is that the amplitude of high-frequency information is reduced. This limits the development potential of super-resolution fluorescence microscopy based on lattice illumination.

[0007] Similar to conventional fringe-modulated two-dimensional structured illumination microscopy (2D-SIM), three-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 modulated 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, and 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, three directions and three phases, a total of nine stripe images are required for reconstruction. Rolling reconstruction can reuse the original image. For stripe structured light illumination, rolling reconstruction is required group by group, because the phases in the same direction need to be matched in direction. If single-frame rolling reconstruction is used, the phase image of this group will look for the phase image of the same direction of the previous group. This causes the original images in the same direction to not be collected at adjacent times, resulting in motion artifacts.

[0018] When using lattice structured light illumination, only seven phase images in one direction are needed, a total of seven images for reconstruction. Rolling reconstruction allows the original image to be reused. For 3I-SIM, single-frame rolling reconstruction can be achieved. The reason is that the acquired phase images do not need to be matched 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, and 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 spectrum 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 OTF attenuation part will be lifted in Wiener deconvolution, so that the obtained image still contains a lot of out-of-focus information. Although increasing the Wiener parameter w can reduce the degree of lifted OTF attenuation to a certain extent, it will still suppress the entire high-frequency part, resulting in a decrease in the signal-to-noise ratio. Summary of the invention

[0026] In view of 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 an implementation method thereof, adopts an adaptive compensation filter instead of OTF attenuation to obtain a super-resolution image with background artifacts removed, and effectively removes artifacts caused by severe background in SIM reconstructed images.

[0027] An 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 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 phase-modulated on the incident parallel light by the beam modulation system, 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 +1-order diffracted lights or three -1-order diffracted lights, 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, and 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 are all radially polarized; 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 incident light of radial polarization 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 optimal; 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. The SIM original images are reconstructed as a group; seven SIM original images are preprocessed, parameter estimated, spectrum separated, spectrum shifted and spectrum 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 amplitude at the offset frequency point is adjusted to make the amplitude at the offset frequency point consistent with 1 / 2 of the optical transfer function OTF of the wide-field image corresponding to the ideal twice super-resolution image, so as to effectively remove background-related artifacts and obtain a super-resolution image with background artifacts removed.

[0029] Furthermore, the present invention also includes a beam expansion and shaping device. The laser after passing through the selection device 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. 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 adopts a polarization beam splitter, a half-wave plate and a liquid crystal spatial light modulator, or adopts a digital microlens array, or adopts a diffractive optical element, or adopts a triangularly arranged or hexagonally arranged grating; adopts 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, 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, so that the incident parallel light is diffracted, and by controlling the hexagonal lattice pattern loaded on the liquid crystal spatial light modulator, 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 liquid crystal spatial light modulator passes through the half-wave plate, and the polarization state becomes horizontal polarization, and is transmitted through the polarization beam splitter; adopts a digital micromirror array: the incident light is regulated by the digital micromirror array, and the diffraction contains different diffraction orders, and the digital micromirror array will not change the polarization state of the incident light. Adopting diffractive optical elements: Diffractive optical elements are passive devices. By changing the distribution state of the optical material therein, the required diffraction order can be achieved. The arrangement of +1-order diffraction light or -1-order diffraction light is triangular, 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 radially distributed. The polarization modulator uses a multi-partition half-wave plate or a multi-partition 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 used to conjugate the focused diffracted light 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, and 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 irradiated on the sample surface is less than 54.7°, the high-frequency information amplitude corresponding to the 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 the 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 the 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 best 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 structure light illumination microscope based on three-beam interference.

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

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

[0044] 2) The linearly polarized light beam is passed through a light beam modulation system to phase modulate the incident parallel light, so that the incident parallel light is diffracted, and the hexagonal lattice pattern formed in the light beam modulation system is controlled so that the diffracted light after phase modulation has at least three +1-order diffracted lights or three -1-order diffracted lights, and the diffracted light emitted from the light 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) Filter out the unnecessary zero-order and extra-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;

[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, and the polarization direction of each diffracted light is accurately controlled by the polarization modulator without losing the laser power. The polarization state of each 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 diffracted light beam passing through the polarization modulator is radial polarization;

[0049] 6) The three radially polarized diffracted lights pass through the 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, and the radially polarized incident light is irradiated on the sample at an incident angle greater than 54.7° to cause interference, and the interference pattern is a hexagonal lattice pattern;

[0051] 8) The fluorescence emitted by the sample is emitted through the same objective lens, and is focused onto the target surface of the camera through a dichroic mirror and a tube lens, and then transmitted to the computer to obtain a 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 optimal;

[0052] 9) 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 SIM original 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 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. 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.

[0055] Wherein, 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. The arrangement of the +1st order diffracted light or the -1st order diffracted light is a triangular arrangement, 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 light beam modulation system so that the diffracted light after phase modulation passes through the space mask and only retains three +1 or three -1 order diffracted lights; the polarization modulation direction of each area of ​​the polarization modulator is different, and the polarization direction of each diffracted light beam is accurately adjusted respectively, and the polarization state of each diffracted light beam 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 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 to enhance the amplitude of high-frequency information; the present invention points out through theoretical analysis that under different extension frequencies of structured light illumination, the corresponding optimal polarization state is different, thereby expanding the applicability of the lattice illumination microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 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 A schematic diagram of the polarization state in the light path of an embodiment of a lattice structure 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 structure 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 structure light illumination microscope based on three-beam interference of the present invention;

[0066] Figure 5 The signal-to-noise ratio curve diagram of an embodiment of the lattice structure 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 structure 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, outputs lasers of multiple wavelengths, and selects the wavelength of the laser and controls the intensity of the laser through the acousto-optic tunable filter; the beam expansion and shaping device uses the first and second lenses and a single-mode polarization-maintaining optical fiber; the first lens focuses the laser into the single-mode polarization-maintaining optical fiber, and the single-mode polarization-maintaining optical 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 optical fiber is vertical polarization. After the laser is output through the single-mode polarization-maintaining optical 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, 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, so that the incident parallel light is diffracted, and 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 the polarization state becomes horizontal polarization, and is transmitted through the polarization beam splitter; the Fourier transform system uses a third lens, and the third lens 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 the 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, and the arrangement of +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-partition 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-partition half-wave plate without losing laser power, so that the polarization state of each beam of diffraction light passing through the three-partition 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 conjugation system adopts a pair of coupling lenses, including a fourth and a fifth lens, and the diffraction light focused by the pair of coupling lenses passes through a 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 interfere, 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 on the target surface of the camera through a polarization-maintaining dichroic mirror and a 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, and a total of seven SIM original images are reconstructed as a group; the seven SIM original images are preprocessed, parameter estimated, spectrum separated, spectrum shifted and spectrum 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 a 1 / 2 scaling 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 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 illumination, the high-frequency information modulation amplitude of lattice illumination is weaker than that of stripe illumination. Therefore, in low signal-to-noise ratio images, the illumination parameters are prone to estimation bias and estimation error, which limits the widespread application of lattice illumination strategies. The present invention uses the method of adjusting the polarization state of the diffracted light to radial polarization, which increases the high-frequency information modulation amplitude by about 50% under high spread spectrum conditions compared to the original angular polarization.

[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 lights 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]

[0081] 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.

[0082] The high frequency modulation amplitude is calculated as:

[0083]

[0084] Through numerical calculation, the result can be obtained:

[0085]

[0086] 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

[0087] (3) Radial polarization

[0088] 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:

[0089]

[0090] 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.

[0091] The high frequency modulation amplitude is calculated as:

[0092]

[0093] Through numerical calculation, it can be further found that when θ>54.7, m rp >1 / 6=m ap , and when m cp <1 / 6=m ap .

[0094] 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.

[0095] Combining the above numerical calculations, we can get the corresponding intensity of the high-frequency information amplitude under 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.

[0096] from Figure 5 The following conclusions can be drawn: when the incident angle of the interference light irradiated on 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 the three cases 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. Therefore, under high spread spectrum conditions, radial polarization is the best choice.

[0097] Finally, it should be noted that the purpose of publishing the embodiments is to help further understand the present invention, but those skilled in the art can understand 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, and the scope of protection claimed by the present invention shall be subject to 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 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 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 phase-modulated on the incident parallel light by the beam modulation system, 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 unnecessary zero-order and redundant-order diffraction light through a 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 laser power, and 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 optimal; 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 original SIM images are reconstructed as a group; seven original SIM images are preprocessed, parameter estimated, spectrum separated, spectrum shifted and spectrum 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 amplitude at the offset frequency point is adjusted to make the amplitude at the offset frequency point consistent with 1 / 2 of the optical transfer function OTF of the wide-field image corresponding to the ideal twice super-resolution image, so as to effectively remove background-related artifacts and obtain 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. The laser beam after passing through the selection device is expanded and mode-shaped by the beam expansion and shaping device, and the 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, characterized in that: The invention comprises 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 adopts a pair of coupling lenses or a pair of concave reflecting mirrors.

4. The lattice structured light illumination microscope according to claim 1, characterized in that: 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 microlens array, or adopts a diffractive optical element.

5. The lattice structured light illumination microscope according to claim 1, characterized in that: 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, characterized in that: The adaptive compensation filter adopts the form of Gaussian filter. 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 lasers of multiple wavelengths, and the wavelength of the laser is selected and the intensity of the laser is controlled by a gating device; 2) The linearly polarized light beam is passed through a light beam modulation system to phase modulate the incident parallel light, so that the incident parallel light is diffracted, and the hexagonal lattice pattern formed in the light beam modulation system is controlled so that the diffracted light after phase modulation has at least three +1-order diffracted lights or three -1-order diffracted lights, and the diffracted light emitted from the light 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) Filter out the unnecessary zero-order and extra-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; 5) The polarization modulator is divided into at least three uniform regions along the radial direction, each region has a different polarization modulation direction, each beam of +1 order diffraction light or -1 order diffraction light is incident on the corresponding region, and the polarization direction of each beam of diffraction light is accurately controlled by the polarization modulator without losing laser power, and 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; 6) The three radially polarized diffracted lights pass through the 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, and the radially polarized incident light is irradiated on the sample at an incident angle greater than 54.7° to cause interference, and the interference pattern is a hexagonal lattice pattern; 8) The fluorescence emitted by the sample is emitted through the same objective lens, 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, and each phase shift obtains a SIM original image, and a total of seven SIM original 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 twice 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 twice super-resolution image, thereby 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, characterized in that: 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, characterized in that: 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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