Nonlinear focus modulation microscopic imaging device and method

Abstract

The invention discloses a nonlinear focus modulation microscopic imaging device and method, and relates to the technical field of microscopic imaging. Comprising a lighting system, a detection system and a computer, the lighting system comprises a laser, an electro-optical modulation module, a phase modulation module and a fluorescence detection module which are arranged in sequence, the laser is used for emitting laser beams with sufficient power to enable response of fluorescence molecules to generate a nonlinear saturation effect, and the electro-optical modulation module rapidly converts the polarization state of the laser beams; the phase modulation module performs phase modulation on the linearly polarized light to form an illumination sample and excite a light spot of a fluorescence signal, and the fluorescence detection module processes the fluorescence signal to obtain a scanning result of a saturated solid light spot and a hollow light spot. An optical system can be simplified, noise possibly brought by sample movement and system instability and artifacts possibly introduced after differential operation are reduced, and the imaging quality and the signal-to-noise ratio are improved.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and in particular to a nonlinear focus modulation microscopic imaging device and method. Background Art

[0002] The traditional nonlinear focus modulation microscopy technology has the problem that the imaging speed is limited and cannot image samples with high time resolution requirements, and motion artifacts may appear during the imaging process. By changing the modulation method of the excitation light and reducing the time required for the conversion of saturated solid and hollow spots, the conversion of saturated solid and hollow spots can be achieved during the scanning process, thereby improving the time resolution and reducing the phenomenon of artifacts that may appear due to sample movement. However, when high-speed modulation is performed, a suitable detection method is required to timely acquire and extract the fluorescence signals obtained by saturated solid and hollow scanning. Traditional nonlinear focus modulation microscopy technology usually uses avalanche photodiodes or photomultiplier tubes as detectors. Although this instrument can detect weak signals as a detector, it also amplifies the noise. The imaging results often have a certain amount of noise. Especially when detecting weak signals, the influence of noise is more obvious, which seriously affects the imaging quality.

[0003] Therefore, it is an urgent problem for those skilled in the art to propose a nonlinear focus modulation microscopic imaging device and method to solve the problems existing in the prior art. Summary of the invention

[0004] In view of this, the present invention provides a nonlinear focus modulation microscopy imaging device and method, which can simplify the optical system, reduce the noise that may be caused by sample movement and system instability and the artifacts that may be introduced after differential operation, and improve imaging quality and signal-to-noise ratio.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A nonlinear focus modulation microscopic imaging device comprises an illumination system, a detection system and a computer, wherein the illumination system comprises a laser, an electro-optical modulation module, a phase modulation module and a fluorescence detection module arranged in sequence;

[0007] A laser, used to emit a laser beam with sufficient power to produce a nonlinear saturation effect on the response of the fluorescent molecules;

[0008] An electro-optic modulation module is used to control the rapid conversion of the laser beam between two different polarization states: horizontal polarization and vertical polarization;

[0009] A phase modulation module performs phase modulation on the linearly polarized light to form saturated solid light spots and hollow light spots for illuminating the sample and exciting the fluorescence signal;

[0010] The fluorescence detection module collects, amplifies, filters and demodulates the fluorescence signal to obtain the scanning results of saturated solid spots and hollow spots.

[0011] Optionally, in the above device, the electro-optic modulation module comprises: an electro-optic modulator for modulating the polarization state of the excitation light and a high-voltage driving unit for controlling the electro-optic modulator, wherein the high-voltage driving unit is controlled by a computer.

[0012] In the above device, optionally, a polarizing plate is provided between the laser and the electro-optical modulation module for adjusting the excitation light to have an angle of 45° with the crystal optical axis direction of the electro-optical modulator, and the adjusted excitation light is horizontally polarized light.

[0013] In the above device, optionally, the excitation light passes through an electro-optic modulator, and when the electro-optic modulator does not apply a control voltage, the polarization state of the excitation light does not change. When the electro-optic modulator applies a half-wave voltage V π When the voltage is large or small, the excitation light is converted into vertically polarized light.

[0014] In the above-mentioned device, optionally, the phase modulation module adopts a polarization-sensitive spatial light modulator. When the excitation light is horizontally polarized light, the excitation light is phase-modulated by the 0 to 2π vortex phase modulation pattern loaded by the phase modulation module when it first reaches the surface of the spatial light modulator to obtain a saturated hollow spot. When the excitation light is vertically polarized light, the spatial light modulator does not perform phase modulation when the excitation light first reaches the surface.

[0015] In the above device, the fluorescence detection module includes a photomultiplier tube for collecting saturated fluorescence signals and a phase-locked amplifier for realizing correlation detection, amplification, filtering and demodulation of fluorescence signals, and the phase-locked amplifier is controlled by a computer.

[0016] In the above-mentioned device, optionally, the reference signal of the phase-locked amplifier is synchronized with the control signal of the electro-optical modulator, and the scanning signals of the saturated solid and hollow spots that change as the electro-optical modulator adjusts the polarization state of the excitation light are obtained by demodulation, and the microscopic imaging results are obtained by post-processing.

[0017] A nonlinear focus modulation microscopic imaging method, used to implement any of the nonlinear focus modulation microscopic imaging devices described above, comprises the following steps:

[0018] S1, the laser emits a laser beam with enough power to make the fluorescent molecules respond to produce a nonlinear saturation effect, and obtain a saturated fluorescence signal;

[0019] S2, after the laser beam is collimated, the electro-optical modulation module controls the rapid conversion of the excitation light between horizontal polarized light and vertical polarized light to obtain saturated solid light spots and hollow light spots;

[0020] S3. The saturated fluorescence signal is amplified by the fluorescence detection module, filtered and demodulated to obtain scanning images of saturated solid light spots and hollow light spots, and the scanning images are subjected to multi-image deconvolution processing to obtain microscopic imaging results.

[0021] It can be seen from the above technical solutions that, compared with the prior art, the present invention provides a nonlinear focus modulation microscopy imaging device and method, which have the following beneficial effects: 1) A simple improvement is made on the basis of the traditional nonlinear focus modulation microscopy imaging system, the system is relatively simple, and is applicable to most fluorescence microscopy imaging systems; 2) No second modulation optical path is introduced, and a single optical path is used to modulate the excitation light, thereby avoiding errors that may be introduced when the adjustment optical paths overlap; 3) The method of using electro-optical modulation technology in combination with a polarization-sensitive spatial light modulator to perform phase modulation to adjust the excitation light to a saturated hollow light spot is easier to implement than phase modulation, and simplifies the optical system; 4) The electro-optical modulation technology is used to realize the rapid conversion of the polarization state of the excitation light, so that the laser can be quickly converted between the saturated solid and hollow states during the scanning process, effectively reducing the noise that may be caused by sample movement and system instability and the artifacts that may be introduced after the differential operation; 5) A phase-locked amplifier is used to realize the correlation detection of the fluorescence signal, filter out the noise and interference signals with different frequencies and phases from the reference signal, i.e., the driving signal of the electro-optical modulator, and improve the imaging quality and signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 A flow chart of a nonlinear focal modulation microscopy imaging method disclosed in the present invention;

[0024] Figure 2 It is a schematic structural diagram of a nonlinear focus modulation microscopic imaging device disclosed in the present invention;

[0025] Figure 3 A schematic diagram of polarization modulation of input light by the electro-optic modulator disclosed in the present invention;

[0026] Figure 4a It is a schematic diagram of the horizontal plane spot distribution of the vertically polarized light disclosed in the present invention;

[0027] Figure 4b It is a schematic diagram of the longitudinal plane spot distribution of the vertically polarized light disclosed in the present invention;

[0028] Figure 4c It is a schematic diagram of the horizontal plane spot distribution of the horizontally polarized light disclosed in the present invention;

[0029] Figure 4d It is a schematic diagram of the longitudinal plane spot distribution of the horizontally polarized light disclosed in the present invention;

[0030] Figure 5 Schematic diagram of the process of multi-view deconvolution algorithm using point spread functions obtained by saturated hollow and solid spot illumination in nonlinear focus modulation microscopy imaging, (a) is the imaging result obtained by illuminating a spoke-shaped sample with an unsaturated solid spot; (b) is the imaging result obtained by illuminating a spoke-shaped sample with a saturated solid spot; (c) is the imaging result obtained by illuminating a spoke-shaped sample with a saturated hollow spot; (d) is the Richardson-Lucy deconvolution result of the imaging result obtained by illuminating a spoke-shaped sample with a saturated solid spot; (e) is the Richardson-Lucy deconvolution result of the imaging result obtained by illuminating a spoke-shaped sample with a saturated hollow spot; (f) is the result obtained by performing multi-view deconvolution processing on the imaging results in (b) and (c);

[0031] Figure 6 Schematic diagram of the process of using a phase-locked amplifier to amplify, demodulate and filter signals in correlation detection, (a) is the distribution diagram of the signal to be detected without the introduction of noise; (b) is the distribution diagram of the signal after the introduction of noise; (c) is the distribution diagram of the reference signal with the same frequency and phase as the signal to be detected; (d) is the signal distribution diagram obtained after correlation detection of the noise signal using the phase-locked amplifier. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Based on the traditional nonlinear focus modulation microscopy imaging system, the present invention uses polarization modulation combined with phase modulation as a method for obtaining saturated solid and hollow light spots. After collimation and beam expansion, the laser first passes through a polarizer whose polarization direction is at an angle of 45° to the crystal optical axis direction of the electro-optical modulator and is adjusted to horizontal linear polarized light, and then enters the electro-optical modulator. Under the control of the electro-optic modulator, the laser realizes rapid conversion between horizontal and vertical linear polarized light at an angle of plus or minus 45° between the polarization direction and the crystal optical axis direction of the electro-optic modulator. After the first phase modulation by the polarization-sensitive spatial light modulator loaded with different phase modulation patterns at different positions, the polarization direction is rotated 90° after being adjusted by a 1 / 4 wave plate twice, and then phase modulated by the spatial light modulator again. The horizontal linear polarized light is converged into a saturated hollow spot after being modulated by the spatial light modulator for the first time and passing through the optical microscopy imaging system. The vertical linear polarized light is converged into a saturated solid spot after being modulated by the spatial light modulator for the second time and passing through the optical microscopy imaging system. The sample is scanned with the saturated solid and hollow spots respectively, and the obtained imaging results are subjected to multi-view deconvolution processing to obtain nonlinear focus modulation microscopy imaging results.

[0034] The electro-optic modulation technology is combined with the phase-locked amplifier detection. The electro-optic modulator is used to realize the rapid conversion of the input light between horizontal and vertical linear polarized light. The light spot obtained after modulation by the polarization-sensitive electro-optic modulator and the optical microscopy imaging system is rapidly converted between a saturated solid light spot and a hollow light spot. During the scanning process, the light spot is rapidly converted between a saturated solid light spot and a hollow light spot. The signal with the same frequency and phase as the driving signal of the electro-optic modulator is used as the reference signal of the phase-locked amplifier. After demodulation, filtering and amplification, the detected noise and interference signals are effectively filtered out, and only the signal with the same frequency and phase as the reference signal is extracted, thereby greatly improving the final imaging quality and signal-to-noise ratio.

[0035] The invention discloses a nonlinear focus modulation microscopic imaging device, comprising an illumination system, a detection system and a computer, wherein the illumination system comprises a laser, an electro-optical modulation module, a phase modulation module and a fluorescence detection module which are arranged in sequence;

[0036] A laser, used to emit a laser beam with sufficient power to produce a nonlinear saturation effect on the response of the fluorescent molecules;

[0037] An electro-optic modulation module is used to control the rapid conversion of the laser beam between two different polarization states: horizontal polarization and vertical polarization;

[0038] A phase modulation module performs phase modulation on the linearly polarized light to form saturated solid light spots and hollow light spots for illuminating the sample and exciting fluorescence;

[0039] The fluorescence detection module collects, amplifies, filters and demodulates the fluorescence signal to obtain the scanning results of saturated solid spots and hollow spots.

[0040] Furthermore, the electro-optic modulation module includes: an electro-optic modulator for modulating the polarization state of the excitation light and a high-voltage driving unit for controlling the electro-optic modulator, and the high-voltage driving unit is controlled by a computer.

[0041] Furthermore, the working principle of the electro-optic modulator is the electro-optic effect, that is, the phenomenon that the refractive index of the electro-optic material changes under the action of a DC or AC electric field. For anisotropic electro-optic materials, the electric field will affect the refractive index distribution of the material in all directions. The Pockels effect refers to the effect that the change in refractive index is proportional to the applied electric field, which can also be called the linear electro-optic effect. Under the action of an external electric field, the refractive index n(E) of the electro-optic material is a function of the external electric field amplitude E, and changes slightly with the change of E, which can be written as: Any polarized light can be decomposed into two orthogonal polarization components. In anisotropic media, these two polarization components are generally decomposed into two principal refractive index axes of the refractive index ellipsoid. Suppose the refractive indices corresponding to the two polarization components are n 1 and n 2 , then their propagation speeds are c 0 / n 1 and c 0 / n 2 , where c 0 is the speed of light waves in a vacuum. Due to the difference in the propagation speed of the two components, when one component propagates to the other end of the device, it will advance or lag behind the other polarization component in time, thus forming a phase delay and changing the polarization state of the outgoing light. When an external electric field E is applied to the anisotropic material, for the Pockels effect, when light passes through a modulator with a length of L, the phase difference between the two polarization components is: For a transverse modulator where the direction of the applied electric field is perpendicular to the propagation direction of light, the electric field E = V / d, where V is the applied voltage and d is the distance between the two electrodes. The expression for the phase difference can be written as: where Γ 0 =k 0 (n 1 -n 2 )L is the phase difference of the device itself, and the half-wave voltage is The phase delay is linearly related to the applied voltage. When the voltage is V π When , the phase delay is π.

[0042] Furthermore, a polarizing plate is provided between the laser and the electro-optical modulation module for adjusting the excitation light to have an angle of 45° with the crystal optical axis direction of the electro-optical modulator, and the adjusted excitation light is horizontally polarized light.

[0043] Furthermore, the excitation light passes through the electro-optic modulator. When the electro-optic modulator does not apply a control voltage, the polarization state of the excitation light does not change. When the electro-optic modulator applies a half-wave voltage V π When the voltage is large or small, the excitation light is converted into vertically polarized light.

[0044] Specifically, the electro-optic modulator applies a voltage with a spacing of V π The high-frequency pulse voltage can realize the rapid conversion of the polarization direction of the excitation light between the horizontal and vertical states.

[0045] Furthermore, the present application adjusts the incident laser to be linearly polarized light whose polarization direction is 45° with the crystal optical axis direction of the electro-optic modulator. In this case, the laser can be regarded as the sum of two components with equal amplitudes and orthogonal polarization directions, whose polarization directions are parallel to the optical axis and perpendicular to the optical axis respectively. When a voltage is applied to make the electro-optic modulator modulate the light beam, the relative phase difference between the two components changes. When the phases of the two components are the same or differ by 180°, the laser is linearly polarized light. Therefore, when the applied voltage is V π When the phase difference between the two components changes by 180°, the two components switch between the same phase or a phase difference of 180°, which is manifested as linear polarized light switching between two states with polarization directions perpendicular to each other.

[0046] Furthermore, the phase modulation module adopts a polarization-sensitive spatial light modulator. When the excitation light is horizontally polarized light, the excitation light is phase-modulated by the vortex phase modulation pattern of 0 to 2π loaded by the phase modulation module when it first reaches the surface of the spatial light modulator, thereby obtaining a saturated hollow light spot. When the excitation light is vertically polarized light, the spatial light modulator does not perform phase modulation when the excitation light first reaches the surface.

[0047] Furthermore, the phase modulation device is a polarization-sensitive spatial light modulator loaded with different phase modulation patterns at different positions.

[0048] After the excitation light is phase modulated by the spatial light modulator for the first time, it is polarized by the 1 / 4 wave plate, and the polarization direction is rotated 90° before reaching the surface of the spatial light modulator for the second phase modulation.

[0049] Furthermore, the fluorescence detection module includes: a photomultiplier tube for collecting saturated fluorescence signals and a phase-locked amplifier for realizing correlation detection, amplifying, filtering and demodulating the fluorescence signals, and the phase-locked amplifier is controlled by a computer.

[0050] Furthermore, in a microscopic imaging system, the intensity distribution of the light spot near the focus of the microscope objective is usually affected by the polarization state, input light spot shape, lens structure and phase plate structure. When a microscope objective with a high numerical aperture is used, the electric field vector distribution near the focus can be expressed as follows according to the vector diffraction theory: in is a point (r 2 ,φ 2 ,z 2 ) is the electric field vector at (r 2 ,φ 2 ,z 2 ) is the cylindrical coordinate distribution of the origin at the focus, C is a normalized constant,

[0051] A 1 (θ, φ) is the amplitude function of the input light, which can usually be expressed as 1 or a deformation of the basic Gaussian function, A 2 (θ, φ) is a 3×3 matrix representing the imaging lens structure, which can usually be expressed as

[0052] p x ;p y ;p z ] is the unit matrix vector representing the polarization state of the input light.

[0053] In a nonlinear focus modulation microscopy imaging system, a phase plate or a spatial light modulator can be used to introduce a phase delay to the input light, thereby changing the energy distribution of the light spot. The electric field vector distribution near the focus can be expressed as: Where Δα(θ,φ) is the introduced phase delay. A vortex phase plate with a range of 0 to 2π is usually used to generate a hollow light spot, and the introduced phase delay can be expressed as Δα=φ. By substituting the above formula, it can be obtained that when the input light is horizontally polarized light, the light spot obtained at the focus after phase modulation by the spatial light modulator is distributed as a hollow light spot in the horizontal plane, and when the input light is vertically polarized light, the light spot obtained at the focus after phase modulation by the spatial light modulator is distributed as a solid light spot in the horizontal plane.

[0054] Furthermore, the reference signal of the phase-locked amplifier is synchronized with the control signal of the electro-optic modulator, and the scanning signals of the saturated solid and hollow spots that change as the electro-optic modulator adjusts the polarization state of the excitation light can be obtained by demodulation, and the microscopic imaging results can be obtained by post-processing.

[0055] Specifically, the phase-locked amplifier is a weak signal correlation detection device based on the cross-correlation reception theory. It can effectively suppress noise using a phase-sensitive detector. It is usually composed of three main parts: a signal channel, a reference channel, and a phase-sensitive detector. The signal channel amplifies the detected signal and narrow-band filters it to output a signal U s The reference channel provides a square wave signal U with the same frequency and adjustable phase as the measured signal through the trigger circuit, frequency multiplication circuit, phase shift circuit and square wave drive circuit. r The phase-sensitive detector consists of a multiplier, a low-pass filter and a DC amplifier. The input signal U s and the reference signal U r The frequency is mixed in the phase-sensitive detector and passed through a low-pass filter to obtain a DC output component proportional to the input signal amplitude. s and the reference signal U r When it is a sine wave: U s =U sm cosωt,U r =U rm cos(ωt+φ). After mixing by the multiplier, the output signal is: Where φ is the input signal U s and the reference signal U r After the input signal and the reference signal are correlated, the spectrum of the output signal is transformed from ω to the frequency band of difference frequency 0 and sum frequency 2ω, where the sum frequency signal component 2ω is filtered out by the low-pass filter, and the output signal of the low-pass filter is: Therefore, the output signal amplitude of the phase-locked amplifier depends on the amplitude of the input signal and the reference signal and the phase difference φ between the two. When φ = 0, That is, the maximum DC output signal can only be obtained when the measured signal and the reference signal have the same frequency and phase in the input signal, and the noise output is an AC signal, which can be filtered out by the low-pass filter connected later. Therefore, the phase-locked amplifier can extract the effective signal from the noise. If the low-pass filter in the phase-sensitive detector uses a first-order RC filter, the transfer function is: The corresponding equivalent noise bandwidth is: Since the measured signal and the reference signal in the phase-locked amplifier are synchronized, there is no problem of frequency stability, so it can be regarded as a tracking filter with high quality Q and strong noise suppression ability. Since the white noise voltage is proportional to the square root of the noise bandwidth, the signal-to-noise improvement ratio of the phase-locked amplifier can be expressed as: Where SNR o is the output signal-to-noise ratio of the lock-in amplifier, SNRi is the input signal-to-noise ratio of the lock-in amplifier, Δf eo is the output noise bandwidth, Δf ei is the noise bandwidth of the input.

[0056] A nonlinear focal modulation microscopy imaging method, such as Figure 1 As shown, a nonlinear focus modulation microscopic imaging device for performing any of the above items comprises the following steps:

[0057] S1, the laser emits a laser beam with enough power to make the fluorescent molecules respond to produce a nonlinear saturation effect, and obtain a saturated fluorescence signal;

[0058] S2, after the laser beam is collimated, the electro-optical modulation module controls the rapid conversion of the excitation light between horizontal polarized light and vertical polarized light to obtain saturated solid light spots and hollow light spots;

[0059] S3. The saturated fluorescence signal is amplified by the fluorescence detection module, filtered and demodulated to obtain scanning images of saturated solid light spots and hollow light spots, and the scanning images are subjected to multi-image deconvolution processing to obtain microscopic imaging results.

[0060] In a specific embodiment, Figure 2 As shown, a single-path modulated fluorescence differential microscopy imaging system device includes: a laser 1, a single-mode optical fiber 2, a collimating lens 3, a polarizer 4, an electro-optic modulator 5, a high-voltage driving module 6, a first reflector 7, a spatial light modulator 8, a 1 / 4 wave plate 9, a first lens 10, a second reflector 11, a third reflector 12, a 1 / 2 wave plate 13, a 1 / 4 wave plate 14, a dichroic mirror 15, a galvanometer scanning system 16, a scanning lens 17, a field lens 18, a fourth reflector 19, a high numerical aperture objective lens 20, a sample 21, a filter 22, a second lens 23, a multimode optical fiber 24, a detector photomultiplier tube 25, a phase-locked amplifier 26, and a computer 27.

[0061] The polarizer 4 is used to adjust the polarization state of the excitation light to a horizontal linear polarized light whose polarization direction is 45° with the crystal optical axis direction of the electro-optical modulator 5 and parallel to the modulation direction of the polarization-sensitive spatial light modulator 8, so as to facilitate the polarization modulation of the electro-optical modulator and the phase modulation of the spatial light modulator; the electro-optical modulator 5 is used to modulate the polarization state of the excitation light to convert it between linear polarized light with an angle of plus or minus 45° with the crystal optical axis direction of the electro-optical modulator, i.e., horizontal and vertical linear polarized light; the polarization-sensitive spatial light modulator 8 is used to modulate the linear polarized light into a hollow light spot or a solid light spot by loading different phase modulation patterns at different positions; the 1 / 4 wave plate 9 is used to adjust the polarization direction of the excitation light; the first lens 10, the second reflector 11 and the third The reflector 12 is used to adjust the optical path; the 1 / 2 wave plate 13 and the 1 / 4 wave plate 14 are used to adjust the polarization state of the modulated excitation light to circularly polarized light; the scanning lens 17 and the field lens 18 form a 4f system, which is used to match the beam size of the excitation light with the numerical aperture of the objective lens 20 and ensure that it is parallel light at the entrance pupil position of the objective lens; the light transmission band of the filter 22 needs to match the fluorescence band of the sample; the reference signal of the phase-locked amplifier 26 needs to have the same frequency and phase as the driving signal of the electro-optical modulator 5; the drive of the galvanometer scanning system 16 needs to match the driving signal of the electro-optical modulator 5. For each imaging unit, after obtaining the imaging results under the two illumination conditions of saturated solid and hollow, the galvanometer scanning system is controlled to scan and image the next imaging unit.

[0062] use Figure 2 The process of realizing nonlinear focal modulation microscopy imaging by the device shown is as follows:

[0063] (1) The excitation light emitted by the laser 1 is output from the single-mode optical fiber 2, collimated and expanded by the collimating lens 3, and then converted into horizontal linear polarized light by the polarization plate 4, the polarization direction of which is 45° with the crystal optical axis direction of the electro-optic modulator 5 and is parallel to the modulation direction of the polarization-sensitive spatial light modulator 8;

[0064] (2) Horizontally polarized light enters the electro-optic modulator 5 for polarization modulation. Under the control of the high voltage driving module 6, when the driving voltage is 0, the excitation light is horizontally polarized light with a polarization direction and an angle of 45° between the crystal optical axis of the electro-optic modulator. When the driving voltage is the half-wave voltage V π When the size is large or small, the excitation light is converted into a vertical linear polarized light with a polarization direction of -45° to the crystal optical axis direction of the electro-optic modulator. As the driving voltage changes, the excitation light quickly switches between the two polarization directions of linear polarized light. The electro-optic modulator can achieve a maximum operating frequency of 8 MHz. At this operating frequency, the excitation light can be converted 8 times in 1 μs, thereby realizing real-time scanning FED imaging.

[0065] (3) After being reflected by the first reflector 7, the linearly polarized light enters the polarization-sensitive spatial light modulator 8 controlled by the computer 27 and loaded with different phase modulation patterns at different positions for phase modulation. When the linearly polarized light is horizontally polarized light, the spatial light modulator performs the first modulation on the laser. After the excitation light is adjusted by the first lens 10 and the second reflector 11, the excitation light whose polarization direction is rotated by 90° after being adjusted by the 1 / 4 wave plate 9 twice reaches the surface of the spatial light modulator again. At this time, the spatial light modulator does not perform the second modulation on the excitation light, and finally obtains a saturated hollow light spot. When the linearly polarized light is vertically polarized light, the spatial light modulator does not perform the first modulation on the laser but only performs the second modulation, and finally obtains a saturated solid light spot. The phase-modulated excitation light is reflected by the third reflector 12 and then passes through the 1 / 2 wave plate 13 and the 1 / 4 wave plate 14 to compensate for the phase difference caused by the third reflector 12, the dichroic mirror 15 behind it, the galvanometer scanning system 16 and the fourth reflector 19, and is adjusted so that the modulated excitation light is circularly polarized light; the excitation light enters the galvanometer scanning system 16 after being reflected by the dichroic mirror 15, and two-dimensional scanning is realized on the final sample surface; the excitation light coming out of the galvanometer scanning system 16 passes through the 4f system composed of the scanning lens 17 and the field lens 18, and the light beam size is adjusted to match the numerical aperture size of the objective lens 20; the excitation light is focused by the objective lens 20 to obtain an excitation light spot limited by the diffraction limit, and the excitation light spot is focused on the fluorescent sample 21;

[0066] (4) The excitation light that switches rapidly between the saturated solid and hollow spot modes illuminates the fluorescent sample 21 and excites the sample to generate a saturated fluorescence signal. The generated saturated fluorescence signal is collected by the objective lens 20 and then passes through the fourth reflector 19, the field lens 18, the scanning lens 17 and the galvanometer scanning system 16 before being incident on the dichroic mirror 15; the dichroic mirror 15 transmits the fluorescence; the transmitted fluorescence is filtered out by the filter 22 to remove the excitation light reflected by the sample and other stray light, and only the fluorescence is emitted; the emitted fluorescence is converged by the second lens 23 and focused on the multimode optical fiber 24, and is transmitted to the detector photomultiplier tube 25 by the multimode optical fiber; the detector photomultiplier tube converts the optical signal into an electrical signal and transmits the electrical signal to the phase-locked amplifier 26;

[0067] (5) The computer 27 controls the electro-optic modulator 5 through the high-voltage driving module 6 to realize the rapid conversion of the excitation light between the solid and hollow illumination modes. The computer 27 controls the reference signal of the phase-locked amplifier 26 to be consistent with the control signal of the high-voltage driving module 6 of the electro-optic modulator 5. The phase-locked amplifier performs correlation detection on the signal detected by the photomultiplier tube to obtain two imaging results corresponding to each scanning imaging unit in the saturated solid or hollow illumination mode. The computer 27 performs multi-view deconvolution processing on the two imaging results to obtain a final image corresponding to a scanning imaging unit.

[0068] (6) The galvanometer scanning system 16 is connected to the computer 27. The computer controls the galvanometer scanning system to perform a two-dimensional scan on the sample to obtain a two-dimensional image corresponding to the sample.

[0069] like Figure 3 As shown in the figure, the change of the voltage applied to the electro-optic modulator will introduce a phase delay between the two polarization states of the input light. For horizontal linear polarized light with a polarization direction of 45° with the crystal optical axis of the electro-optic modulator, when the driving voltage of the electro-optic modulator is equal to the half-wave voltage V of the electro-optic modulator, π When the driving voltage is 0, the phase delay of π will be introduced between the two polarization states of the input light, and the polarization direction of the linear polarized light will be transformed into a vertical linear polarized light with an angle of -45° with the crystal optical axis of the electro-optic modulator. When the driving voltage is 0, the phase delay between the two polarization states of the input light is 0, and the polarization direction of the linear polarized light is a horizontal linear polarized light with an angle of 45° with the crystal optical axis of the electro-optic modulator. π The high-frequency pulse voltage can realize the rapid conversion of the light beam between the horizontal and vertical linear polarized light states with the polarization direction of 45° and -45° to the crystal optical axis of the electro-optic modulator.

[0070] When the input light is vertical linear polarized light whose polarization direction is perpendicular to the modulation direction of the polarization-sensitive spatial light modulator, the spatial light modulator modulates the vertical linear polarized light, and a saturated solid spot is obtained after being converged by the optical system. The specific distribution of the saturated solid spot near the focus of the optical system is calculated according to the vector diffraction theory. The distribution of the saturated solid spot in the horizontal plane is shown in FIG4(a), and the distribution in the longitudinal plane is shown in FIG4(b). When the input light is horizontal linear polarized light whose polarization direction is parallel to the modulation direction of the polarization-sensitive spatial light modulator, the spatial light modulator modulates the horizontal linear polarized light, and a saturated hollow spot is obtained after being converged by the optical system. The specific distribution of the saturated hollow spot near the focus of the optical system is calculated according to the vector diffraction theory. The distribution of the saturated hollow spot in the horizontal plane is shown in FIG4(c), and the distribution in the longitudinal plane is shown in FIG4(d).

[0071] like Figure 5 As shown in the figure, the process of multi-view deconvolution algorithm using the point spread function obtained by saturated hollow and solid spot illumination in nonlinear focus modulation microscopy imaging. The imaging result obtained by illuminating the spoke-shaped sample with the non-saturated solid spot obtained after the non-saturated vertical linear polarized light modulated by the spatial light modulator is converged by the optical system is shown in Figure 5 As shown in (a); the saturated vertical linear polarized light modulated by the spatial light modulator is converged by the optical system to obtain a saturated solid spot to illuminate the spoke-shaped sample. The imaging result is as follows Figure 5As shown in (b); the saturated horizontal linear polarized light modulated by the spatial light modulator is converged by the optical system to obtain a saturated hollow spot to illuminate the spoke-shaped sample. The imaging result is as follows Figure 5 As shown in (c) Figure 5 The result of Richardson-Lucy deconvolution of the imaging result in (b) is as follows Figure 5 As shown in (d) Figure 5 The result of Richardson-Lucy deconvolution of the imaging result in (c) is as follows Figure 5 As shown in (e) in 5; the imaging results in (b) in 5 and (c) in 5 are subjected to multi-view deconvolution processing, and the final imaging result is as follows Figure 5 As shown in (f) in .

[0072] like Figure 6 As shown in FIG. 1 , a phase-locked amplifier with a reference signal having the same frequency and phase as the signal to be detected is used to perform correlation detection on a signal with a certain amount of noise to filter out the noise. The signal to be detected is a cosine signal that changes with time. Figure 6 As shown in (a), the distribution of the detected signal after a certain amount of Gaussian noise is introduced into it over time is as follows Figure 6 As shown in (b), the signal containing noise detected by ordinary detectors is as follows Figure 6 As shown in (b) of the figure, when the reference signal of the phase-locked amplifier is a cosine signal with the same frequency and phase as the signal to be detected, Figure 6 As shown in (c) of FIG. 1 , the detection signal obtained by the phase-locked amplifier after correlation detection is basically consistent with the frequency and phase distribution of the signal to be detected after noise is filtered out. Figure 6 As shown in (d) in .

[0073] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can refer to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system or system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can refer to the partial description of the method embodiment. The system and system embodiments described above are merely schematic, wherein the units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. Ordinary technicians in this field can understand and implement it without creative work.

[0074] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nonlinear focus modulation microscopic imaging device, comprising an illumination system, a detection system and a computer, characterized in that: The illumination system includes a laser, an electro-optical modulation module, a phase modulation module and a fluorescence detection module which are arranged in sequence; A laser, used to emit a laser beam with sufficient power to produce a nonlinear saturation effect on the response of the fluorescent molecules; An electro-optic modulation module is used to control the rapid conversion of the laser beam between two different polarization states: horizontal polarization and vertical polarization; A phase modulation module performs phase modulation on the linearly polarized light to form saturated solid light spots and hollow light spots for illuminating the sample and exciting the fluorescence signal; The fluorescence detection module collects, amplifies, filters and demodulates the fluorescence signal to obtain the scanning results of saturated solid spots and hollow spots.

2. A nonlinear focus modulation microscopic imaging device according to claim 1, characterized in that: The electro-optic modulation module comprises: an electro-optic modulator for modulating the polarization state of the excitation light and a high voltage driving unit for controlling the electro-optic modulator, wherein the high voltage driving unit is controlled by a computer.

3. A nonlinear focus modulation microscopic imaging device according to claim 1, characterized in that: A polarizing plate is provided between the laser and the electro-optical modulation module for adjusting the excitation light to have an angle of 45° with the crystal optical axis direction of the electro-optical modulator, and the adjusted excitation light is horizontally polarized light.

4. A nonlinear focus modulation microscopic imaging device according to claim 3, characterized in that: The excitation light passes through the electro-optic modulator. When the electro-optic modulator does not apply a control voltage, the polarization state of the excitation light does not change. When the electro-optic modulator applies a half-wave voltage V π When the voltage is large or small, the excitation light is converted into vertically polarized light.

5. The nonlinear focus modulation microscopic imaging device according to claim 1, characterized in that: The phase modulation module adopts a polarization-sensitive spatial light modulator. When the excitation light is horizontally polarized light, the excitation light is phase-modulated by the 0 to 2π vortex phase modulation pattern loaded by the phase modulation module when it first reaches the surface of the spatial light modulator, and a saturated hollow light spot is obtained. When the excitation light is vertically polarized light, the spatial light modulator does not perform phase modulation when the excitation light first reaches the surface.

6. The nonlinear focus modulation microscopic imaging device according to claim 1, characterized in that: The fluorescence detection module includes: a photomultiplier tube for collecting saturated fluorescence signals and a phase-locked amplifier for realizing correlation detection, amplification, filtering and demodulation of fluorescence signals, and the phase-locked amplifier is controlled by a computer.

7. A nonlinear focus modulation microscopic imaging device according to claim 6, characterized in that: The reference signal of the phase-locked amplifier is synchronized with the control signal of the electro-optic modulator. The scanning signals of the saturated solid and hollow spots that change as the electro-optic modulator adjusts the polarization state of the excitation light are obtained by demodulation, and the microscopic imaging results are obtained by post-processing.

8. A nonlinear focus modulation microscopy imaging method, used to implement a nonlinear focus modulation microscopy imaging device as claimed in any one of claims 1 to 7, characterized in that: The following steps are involved: S1, the laser emits a laser beam with enough power to make the fluorescent molecules respond to produce a nonlinear saturation effect, and obtain a saturated fluorescence signal; S2, after the laser beam is collimated, the electro-optical modulation module controls the rapid conversion of the excitation light between horizontal polarized light and vertical polarized light to obtain saturated solid light spots and hollow light spots; S3. The saturated fluorescence signal is amplified by the fluorescence detection module, filtered and demodulated to obtain scanning images of saturated solid light spots and hollow light spots, and the scanning images are subjected to multi-image deconvolution processing to obtain microscopic imaging results.

Images