Nonlinear focal modulation microscopy apparatus and method

Abstract

The application discloses a kind of nonlinear focal point modulation microscopic imaging device and method, it is related to microscopic imaging technical field.The illumination system, detection system and computer are included, illumination system includes the laser, electro-optic modulation module, phase modulation module and fluorescence detection module arranged in sequence, laser is used to send laser beam with enough power to make the response of fluorescent molecule produce nonlinear saturation effect, electro-optic modulation module rapidly converts the polarization state of laser beam, phase modulation module carries out phase modulation to linearly polarized light, forms the light spot of illumination sample and excitation fluorescence signal, fluorescence detection module, to fluorescent signal is handled to obtain the scanning result of saturated solid light spot and hollow light spot.The application can simplify optical system, reduce noise and artifact possibly introduced after differential operation possibly brought by sample movement and system instability, improve imaging quality and signal-to-noise ratio.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microscopic imaging technology, in particular to a nonlinear focal modulation microscopic imaging device and method. BACKGROUND

[0002] The traditional nonlinear focal modulation microscopic imaging technology has the problems of limited imaging speed and motion artifacts in the imaging process. By changing the modulation method of the excitation light and reducing the time required for saturated solid and hollow spot conversion, the conversion of saturated solid and hollow spots during scanning can be realized to improve the time resolution and reduce the artifacts that may occur due to sample motion. However, when high-speed modulation is performed, a suitable detection method is needed to timely acquire and extract the fluorescence signals obtained by saturated solid and hollow scanning. The traditional nonlinear focal modulation microscopic imaging technology usually uses avalanche photodiodes or photomultiplier tubes as detectors. Although this instrument can detect weak signals, it also amplifies noise, and the imaging results often have certain noise, especially when detecting weak signals, the influence of noise is more obvious, which seriously affects the imaging quality.

[0003] Therefore, a nonlinear focal modulation microscopic imaging device and method are proposed to solve the problems existing in the prior art, which is a problem that needs to be solved by those skilled in the art. SUMMARY

[0004] Therefore, the present application provides a nonlinear focal modulation microscopic imaging device and method, which can simplify the optical system, reduce noise and artifacts that may be introduced after differential operation caused by sample motion and system instability, and improve imaging quality and signal-to-noise ratio.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

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

[0007] The laser is used to emit a laser beam with sufficient power to make the response of the fluorescent molecules produce nonlinear saturation effect;

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

[0009] The phase modulation module modulates the linearly polarized light to form saturated solid and hollow spots for illuminating the sample and exciting fluorescence signals;

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

[0011] The device, optionally, the electro-optical modulation module comprises an electro-optical modulator for modulating the polarization state of the excitation light and a high-voltage driving unit for controlling the electro-optical modulator, and the high-voltage driving unit is controlled by the computer.

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

[0013] The device, optionally, the excitation light passes through the electro-optical modulator, and when the electro-optical modulator does not apply a control voltage, the polarization state of the excitation light does not change, and when the electro-optical modulator applies a voltage with a size of half-wave voltage V π of the electro-optical modulator, the excitation light is converted into vertically polarized light.

[0014] The 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 phase modulation module loaded with a vortex phase modulation pattern of 0 to 2π when it reaches the surface of the spatial light modulator for the first time, to obtain a saturated hollow light spot, and when the excitation light is vertically polarized light, the spatial light modulator does not perform phase modulation when the excitation light reaches the surface for the first time.

[0015] The device, the fluorescence detection module comprises a photomultiplier for collecting saturated fluorescence signals and a lock-in amplifier for realizing correlation detection, amplification, filtering and demodulation of the fluorescence signal, and the lock-in amplifier is controlled by the computer.

[0016] The device, optionally, the reference signal of the lock-in amplifier and the control signal of the electro-optical modulator are synchronized, the scanning signals of the saturated solid light spot and the hollow light spot that change with the polarization state of the excitation light adjusted by the electro-optical modulator are obtained by demodulation, and the microscopic imaging results are obtained by post-processing.

[0017] A nonlinear focal point modulation microscopic imaging method for performing the nonlinear focal point modulation microscopic imaging device of any one of the above, comprising the following steps:

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

[0019] S2, the collimated laser beam passes through the electro-optical modulation module to control the rapid conversion of the excitation light between horizontally polarized light and vertically polarized light, to obtain a saturated solid light spot and a hollow light spot;

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

[0021] Compared with the prior art, the nonlinear focal point modulation microscopic imaging device and method provided by the application have the following advantages: 1) the device is improved on the basis of the traditional nonlinear focal point modulation microscopic imaging system, the system is relatively simple, and is suitable for most fluorescence microscopic imaging systems; 2) the modulation of excitation light is realized by using a single light path without introducing a second modulation light path, thereby avoiding errors that may be introduced when the light paths are overlapped; 3) the excitation light is adjusted to a saturated hollow light spot by using the phase modulation of the electro-optical modulation technology combined with the polarization-sensitive spatial light modulator, and the method is easier to realize compared with phase modulation, and the optical system is simplified; 4) the polarization state of the excitation light is quickly converted by using the electro-optical modulation technology, the laser is quickly converted between the saturated solid state and the hollow state in the scanning process, and noise and artifacts that may be introduced after differential operation due to sample movement and system instability are effectively reduced; and 5) the correlation detection of the fluorescence signal is realized by using the lock-in amplifier, noise and interference signals of different frequencies and different phases of the filter and the reference signal, i.e., the driving signal of the electro-optical modulator, are filtered out, and the imaging quality and the signal-to-noise ratio are improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of the provided drawings.

[0023] Figure 1 A flow chart of the nonlinear focal point modulation microscopic imaging method disclosed by the application;

[0024] Figure 2 A structural schematic diagram of the nonlinear focal point modulation microscopic imaging device disclosed by the application;

[0025] Figure 3 A schematic diagram of the polarization modulation of the input light by the electro-optical modulator disclosed by the application;

[0026] Figure 4a A schematic diagram of the horizontal plane light spot distribution of the vertical polarization light disclosed by the application;

[0027] Figure 4b A schematic diagram of the longitudinal plane light spot distribution of the vertical polarization light disclosed by the application;

[0028] Figure 4c A horizontal polarization light horizontal plane spot distribution schematic diagram disclosed by the present application;

[0029] Figure 4d A horizontal polarization light longitudinal plane spot distribution schematic diagram disclosed by the present application;

[0030] Figure 5 A process schematic diagram of a multi-view deconvolution algorithm using a point spread function obtained by saturated hollow and solid spot illumination in nonlinear focal modulation microscopic imaging, (a) is an imaging result diagram of a striped sample obtained by non-saturated solid spot illumination; (b) is an imaging result diagram of a striped sample obtained by saturated solid spot illumination; (c) is an imaging result diagram of a striped sample obtained by saturated hollow spot illumination; (d) is a Richardson-Lucy deconvolution result diagram of the imaging result of the striped sample obtained by saturated solid spot illumination; (e) is a Richardson-Lucy deconvolution result diagram of the imaging result of the striped sample obtained by saturated hollow spot illumination; (f) is a result diagram obtained by multi-view deconvolution processing on the imaging results in (b) and (c);

[0031] Figure 6 A process schematic diagram of using a lock-in amplifier to realize amplification, demodulation and filtering of a signal in correlation detection, (a) is a signal distribution diagram without introducing noise; (b) is a signal distribution diagram after introducing noise; (c) is a reference signal distribution diagram with the same frequency and phase as the signal to be detected; (d) is a signal distribution diagram obtained after correlation detection of the noise signal by using a lock-in amplifier. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0033] The application is based on the traditional nonlinear focal point modulation microscopic imaging system, and polarization modulation and phase modulation are combined to obtain saturated solid and hollow light spots, collimated and expanded laser first passes through a polarizer with an angle of 45 degrees between the polarization direction and the crystal optical axis direction of the electro-optical modulator to be adjusted to horizontal linearly polarized light, and then enters the electro-optical modulator. The laser is controlled by the electro-optical modulator to realize the rapid conversion between the horizontal and vertical linearly polarized light with an angle of 45 degrees between the polarization direction and the crystal optical axis direction of the electro-optical modulator. After the first phase modulation of the polarization-sensitive spatial light modulator loaded with different phase modulation patterns at different positions, the polarization direction is turned by 90 degrees after the adjustment of the 1 / 4 wave plate twice, and the horizontal linearly polarized light is modulated by the spatial light modulator again. After the optical microscopic imaging system, the saturated hollow light spot is converged, and the vertical linearly polarized light is modulated by the spatial light modulator again. After the optical microscopic imaging system, the saturated solid light spot is converged. The saturated solid and hollow light spots are used to scan the sample respectively, and the imaging results obtained are processed by multi-view deconvolution to obtain the nonlinear focal point modulation microscopic imaging results.

[0034] The electro-optical modulation technology and the lock-in amplifier detection are combined, the input light is rapidly converted between the horizontal and vertical linearly polarized light by the electro-optical modulator, and the light spot obtained after the polarization-sensitive electro-optical modulator modulation and the optical microscopic imaging system is rapidly converted between the saturated solid light spot and the hollow light spot. The rapid conversion between the saturated solid and hollow light spots is realized in the scanning process. The signal with the same frequency and phase as the driving signal of the electro-optical modulator is used as the reference signal of the lock-in amplifier, the demodulated, filtered and amplified noise and interference signals are effectively filtered out, and only the signal with the same frequency and phase as the reference signal is extracted, so that the final imaging quality and signal-to-noise ratio are greatly improved.

[0035] The application discloses a nonlinear focal point modulation microscopic imaging device, which comprises an illumination system, a detection system and a computer.

[0036] The laser is used for emitting a laser beam with a power sufficient to make the response of a fluorescent molecule produce a nonlinear saturation effect.

[0037] The electro-optical modulation module is used for controlling the rapid conversion of the laser beam between the horizontal polarization light and the vertical polarization light.

[0038] The phase modulation module performs phase modulation on linearly polarized light to form saturated solid and hollow light spots for illuminating a sample and exciting fluorescence.

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

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

[0041] Further, the electro-optical modulator works on the principle of electro-optical effect, i.e. the phenomenon that the refractive index of electro-optical material changes under the action of direct current or alternating current electric field. For anisotropic electro-optical material, the electric field will affect the refractive index distribution of the material in each direction. Pockels effect refers to the effect that the change of refractive index is proportional to the applied electric field, which can also be called linear electro-optical effect. Under the action of external electric field, the refractive index n(E) of the electro-optical material is a function of the amplitude E of the external electric field, and changes slightly with the change of E, which can be written as: Any polarized light can be decomposed into two orthogonal polarization components, and in anisotropic medium, the two polarization components are generally decomposed into two principal refractive index axes of the refractive index ellipsoid. Let the refractive indices corresponding to the two polarization components be n1 and n2, respectively, and the propagation speeds of the two polarization components be c0 / n1 and c0 / n2, respectively, where c0 is the speed of light in vacuum. Due to the difference in propagation speed of the two components, one component will lead or lag the other polarization component in time when it propagates to the other end of the device, thereby 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 Pockels effect, the phase difference generated by the two polarization components when the light passes through the modulator with a length of L is: For a transverse modulator with the direction of the applied electric field perpendicular to the direction of light propagation, the electric field E = V / d, where V is the applied voltage and d is the distance between the two electrodes. The expression of the phase difference can be written as: where Γ0 = k0(n1-n2)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, and when the voltage is V π , the phase delay is π.

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

[0043] Further, the excitation light passes through the electro-optical modulator, and when the electro-optical modulator does not apply a control voltage, the polarization state of the excitation light does not change, and when the electro-optical modulator applies a voltage with a size of half-wave voltage V π of the electro-optical modulator, the excitation light is converted into vertically polarized light.

[0044] Specifically, the electro-optical modulator realizes the fast conversion of the polarization direction of the excitation light between the horizontal and vertical states by applying a high-frequency pulse voltage with a size of V π .

[0045] Further, the incident laser light is adjusted to linearly polarized light with an angle of 45° between the polarization direction and the optical axis direction of the crystal of the electro-optical modulator, in which case the laser light can be regarded as the sum of two components with equal amplitudes and orthogonal polarization directions parallel and perpendicular to the optical axis, respectively. When the voltage is applied to make the electro-optical 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 light is linearly polarized light. Therefore, when the voltage size is V π , the phase difference between the two components changes by 180°, and the conversion between the two states of the same phase or a phase difference of 180° is manifested as the conversion of linearly polarized light between the two states with perpendicular polarization directions.

[0046] Further, the phase modulation module uses 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, 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] Further, 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 passes through the phase modulation of the spatial light modulator for the first time, it passes through the polarization adjustment of the 1 / 4 wave plate, and the polarization direction is turned by 90° before reaching the surface of the spatial light modulator again for the second time.

[0049] Further, the fluorescence detection module includes a photomultiplier tube for collecting saturated fluorescence signals and a lock-in amplifier for realizing correlation detection, amplification, filtering and demodulation of the fluorescence signals, the lock-in amplifier being controlled by a computer.

[0050] Further, in the microscopic imaging system, the intensity distribution of the light spot near the focal point of the microscope objective is usually affected by the polarization state, the input light spot shape, the lens structure and the phase plate structure. When a high numerical aperture microscope objective is used, according to the vector diffraction theory, the electric field vector distribution near the focal point can be expressed as: wherein is the electric field vector at point (r2, φ2, z2), (r2, φ2, z2) is the cylindrical coordinate distribution with the origin at the focal point, C is a normalized constant,

[0051] A1(θ, φ) is the amplitude function of the input light, which can be represented as 1 or a deformation of the basic Gaussian function, A2(θ, φ) is a 3x3 matrix representing the structure of the imaging lens, which can be represented as

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

[0053] In the nonlinear focal modulation microscopic imaging system, the phase delay can be introduced to the input light by using a phase plate or a spatial light modulator, so as to change the energy distribution of the light spot, and the electric field vector distribution near the focal point can be represented as: where Δα(θ, φ) is the introduced phase delay. A vortex phase plate with 0 to 2π is usually used to generate a hollow light spot, and the introduced phase delay can be represented as Δα = φ. By substituting the above formula, it can be obtained that when the input light is horizontally linearly polarized light, the distribution of the light spot in the horizontal plane at the focal point after the phase modulation of the spatial light modulator is a hollow light spot, and when the input light is vertically linearly polarized light, the distribution of the light spot in the horizontal plane at the focal point after the phase modulation of the spatial light modulator is a solid light spot.

[0054] Further, the reference signal of the lock-in amplifier and the control signal of the electro-optical modulator are synchronized, the scanning signal of the saturated solid and hollow light spot which changes with the adjustment of the polarization state of the excitation light by the electro-optical modulator can be obtained by demodulation, and the microscopic imaging result can be obtained by post-processing.

[0055] Specifically, the lock-in amplifier is a weak signal correlation detection device based on the cross-correlation receiving theory, which can effectively suppress noise by using a phase-sensitive detector, and is usually composed of a signal channel, a reference channel and a phase-sensitive detector. The signal channel amplifies and narrows the band of the detected signal and outputs the signal U s , the reference channel provides a square wave signal U r with the same frequency and adjustable phase as the measured signal through a trigger circuit, a frequency multiplication circuit, a phase shift circuit and a square wave driving circuit, and the phase-sensitive detector is composed of a multiplier, a low-pass filter and a DC amplifier. The input signal U s and the reference signal U r are mixed in the phase-sensitive detector, and a direct current output component proportional to the amplitude of the input signal is obtained after the low-pass filter. When the input signal U s and the reference signal U r are sine waves: Us = U sm cosωt, U r = U rm cos(ωt+φ). After mixing in the multiplier, the output signal is: where φ is the phase difference between the input signal U s and the reference signal U r . After correlation operation of the input signal and the reference signal, the frequency spectrum of the output signal is transformed from ω to the frequency band of difference frequency 0 and sum frequency 2ω, wherein the sum frequency signal component 2ω is filtered out by a 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 amplitudes of the input signal and the reference signal and the phase difference φ between the two. When φ = 0, that is, only when the measured signal and the reference signal have the same frequency and the same phase in the input signal, the maximum direct current output signal can be obtained, and the noise output is an alternating current signal, which can be filtered out by the low-pass filter connected after the phase-sensitive detector, so that the phase-locked amplifier can extract the effective signal from the noise. If a first-order RC filter is used for the low-pass filter in the phase-sensitive detector, 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 number Q, which has strong noise suppression capability. 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 phase-locked amplifier, SNR i is the input signal-to-noise ratio of the phase-locked amplifier, Δf eo is the output noise bandwidth, and Δf ei is the input noise bandwidth.

[0056] A nonlinear focal modulation microscopic imaging method, as shown in Figure 1 , is used to perform the nonlinear focal modulation microscopic imaging device of any one of the above, comprising the following steps:

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

[0058] S2, the laser beam is collimated and passes through an electro-optical modulation module to control the rapid conversion of the excitation light between horizontal polarized light and vertical polarized light, and a saturated solid spot and a hollow spot are obtained;

[0059] S3. Amplify the saturated fluorescence signal through the fluorescence detection module, filter and demodulate to obtain scanning images of saturated solid spots and hollow spots, and perform multi-image deconvolution processing on the scanning images 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-optical 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 lock-in 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 at an angle of 45° to the crystal optical axis 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 so that it can be converted between linear polarized light with an angle of plus or minus 45° to the crystal optical axis 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 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 be at 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 saturated solid and hollow illumination conditions, the galvanometer scanning system is controlled to scan and image the next imaging unit.

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

[0063] (1) The excitation light emitted by the laser 1 is output by a single-mode optical fiber 2, collimated and expanded by a collimating lens 3, and then becomes horizontally polarized light with a polarization direction at an angle of 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 after passing through a polarizer 4;

[0064] (2) The horizontally polarized light enters the electro-optical modulator 5 for polarization modulation, and 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 at an angle of 45° with the crystal optical axis direction of the electro-optical modulator, when the driving voltage is the half-wave voltage V π , the excitation light becomes vertically polarized light with a polarization direction at an angle of -45° with the crystal optical axis direction of the electro-optical modulator, and as the driving voltage changes, the excitation light rapidly converts between the two polarization directions of linearly polarized light, and the electro-optical modulator can achieve a working frequency of 8 MHz, which can convert the excitation light 8 times in 1 μs, thereby realizing real-time scanning FED imaging;

[0065] (3) The linearly polarized light is reflected by the first mirror 7 and 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 modulates the laser light for the first time, and after the adjustment of the first lens 10 and the second mirror 11, the excitation light whose polarization direction has been turned by 90° by the adjustment of the 1 / 4 wave plate 9 reaches the surface of the spatial light modulator again, at this time the spatial light modulator does not modulate the excitation light for the second time, and finally a saturated hollow light spot is obtained. When the linearly polarized light is vertically polarized light, the spatial light modulator does not modulate the laser light for the first time but only for the second time, and finally a saturated solid light spot is obtained. After the excitation light modulated in phase is reflected by the third mirror 12, it 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 mirror 12, the dichroic mirror 15, the galvanometer scanning system 16, and the fourth mirror 19 behind it and adjust the modulated excitation light to be circularly polarized light. After the excitation light is reflected by the dichroic mirror 15, it enters the galvanometer scanning system 16, which realizes two-dimensional scanning on the final sample surface. The excitation light from the galvanometer scanning system 16 passes through the 4f system composed of the scanning lens 17 and the field lens 18 to adjust the beam size to match the numerical aperture size of the objective lens 20. After the excitation light passes through the objective lens 20, a diffraction-limited excitation light spot is obtained, and the excitation light spot is focused on the fluorescent sample 21;

[0066] (4) The saturated fluorescence signal generated after the sample is excited by the excitation light illumination of the fluorescence sample 21 in the fast conversion of the saturated solid and hollow spot modes is collected by the objective lens 20, and then enters the dichroic mirror 15 after passing through the fourth mirror 19, the field lens 18, the scanning lens 17 and the galvanometer scanning system 16; the dichroic mirror 15 transmits the fluorescence; the transmitted fluorescence is filtered 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 focused to the multimode optical fiber 24 after being converged by the second lens 23, and then is transmitted to the photomultiplier tube 25 by the multimode optical fiber; the photomultiplier tube converts the optical signal into an electrical signal and transmits the electrical signal to the lock-in amplifier 26;

[0067] (5) The computer 27 controls the fast conversion of the excitation light in the solid and hollow illumination modes by the high-voltage driving module 6 of the electro-optical modulator 5, the computer 27 controls the reference signal of the lock-in amplifier 26 to be consistent with the control signal of the high-voltage driving module 6 of the electro-optical modulator 5, the lock-in amplifier detects the signal detected by the photomultiplier tube, and obtains two imaging results corresponding to each scanning imaging unit in the saturated solid or hollow illumination mode, and the computer 27 obtains the final image corresponding to each scanning imaging unit after multi-view deconvolution processing of the two imaging results;

[0068] (6) The galvanometer scanning system 16 is connected with the computer 27, and the galvanometer scanning system is controlled by the computer to perform two-dimensional scanning on the sample, so as to obtain the two-dimensional image corresponding to the sample.

[0069] As shown in Figure 3 , the change of the voltage applied to the electro-optical modulator will introduce a change in the phase delay between the two polarization states of the input light. For horizontally polarized light with an angle of 45° between the polarization direction and the crystal optical axis of the electro-optical modulator, when the driving voltage of the electro-optical modulator is half the wave voltage V π of the electro-optical modulator, a phase delay of π will be introduced between the two polarization states of the input light, and the polarization direction of the linearly polarized light will change to vertically polarized light with an angle of -45° between the polarization direction and the crystal optical axis of the electro-optical 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 linearly polarized light is horizontally polarized light with an angle of 45° between the polarization direction and the crystal optical axis of the electro-optical modulator. By applying a high-frequency pulse voltage with a size of V π , the fast conversion of the light beam between the two states of horizontally and vertically polarized light with an angle of 45° and -45° between the polarization direction and the crystal optical axis of the electro-optical modulator can be realized.

[0070] When the input light is vertical linearly polarized light with the polarization direction perpendicular to the modulation direction of the polarization-sensitive spatial light modulator, the spatial light modulator modulates the vertical linearly polarized light, and after converging by the optical system, a saturated solid light spot is obtained. According to the vector diffraction theory, the specific distribution of the saturated solid light spot near the focal point of the optical system is calculated. The distribution of the saturated solid light spot in the horizontal plane is shown in Fig. 4(a), and the distribution in the longitudinal plane is shown in Fig. 4(b). When the input light is horizontal linearly polarized light with the polarization direction parallel to the modulation direction of the polarization-sensitive spatial light modulator, the spatial light modulator modulates the horizontal linearly polarized light, and after converging by the optical system, a saturated hollow light spot is obtained. According to the vector diffraction theory, the specific distribution of the saturated hollow light spot near the focal point of the optical system is calculated. The distribution of the saturated hollow light spot in the horizontal plane is shown in Fig. 4(c), and the distribution in the longitudinal plane is shown in Fig. 4(d),

[0071] As shown in Figure 5 , the process of the multi-view deconvolution algorithm using the point spread function obtained by saturated hollow and solid light spot illumination in nonlinear focal modulation microscopic imaging, the non-saturated vertical linearly polarized light modulated by the spatial light modulator converges by the optical system to obtain a non-saturated solid light spot. The imaging result of the radial stripe sample illuminated by the non-saturated solid light spot is shown in (a) of Figure 5 ; the saturated vertical linearly polarized light modulated by the spatial light modulator converges by the optical system to obtain a saturated solid light spot. The imaging result of the radial stripe sample illuminated by the saturated solid light spot is shown in (b) of Figure 5 ; the saturated horizontal linearly polarized light modulated by the spatial light modulator converges by the optical system to obtain a saturated hollow light spot. The imaging result of the radial stripe sample illuminated by the saturated hollow light spot is shown in (c) of Figure 5 ; the Richard-son-Lucy deconvolution of the imaging result in (b) of Figure 5 is shown in (d) of Figure 5 ; the Richardson-Lucy deconvolution of the imaging result in (c) of Figure 5 is shown in (e) of Figure 5 ; the multi-view deconvolution processing of the imaging results in (b) of 5 and (c) of 5 is performed, and the final imaging result is shown in (f) of Figure 6 .

[0072] As shown in Figure 6 , the result of the correlation detection of a signal with noise by a phase-locked amplifier with a reference signal having the same frequency and phase as the signal to be detected, the signal to be detected is a cosine signal changing with time as shown in (a) of Figure 6 , and the distribution of the signal to be detected after introducing a certain Gaussian noise changes with time as shown in Figure 6(b) of FIG. 1 shows a signal containing noise detected by a common detector Figure 6 (b) of FIG. 1 shows a signal containing noise detected by a common detector Figure 6 (c) of FIG. 1 shows a signal detected by a phase-locked amplifier ​ (d) of FIG. 1 shows a signal detected by a phase-locked amplifier

[0073] Each of the embodiments described in the specification is described in a progressive manner, and the same or similar parts among the embodiments can be referred to each other. Each of the embodiments mainly describes the difference from other embodiments. In particular, the system or system embodiment is described simply because it is basically similar to the method embodiment, and the relevant part can be referred to the part of the method embodiment. The system and system embodiment described above are merely illustrative, and the units described as separate components can be or can not be physically separated, and the components shown as units can be or can not be physical units, i.e., they can be located in one place or distributed to multiple network units. Part or all of the modules can be selected to achieve the purpose of the embodiment according to the actual needs. Those skilled in the art can understand and implement it without creative labor.

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

Claims

1. A non-linear focal modulation microscopy apparatus comprising an illumination system, a detection system and a computer, characterized in that, The illumination system comprises a laser, an electro-optical modulation module, a phase modulation module and a fluorescence detection module arranged in sequence. The laser is used to emit a laser beam with a power sufficient to cause a nonlinear saturation effect in the response of the fluorescent molecules. The electro-optical modulation module is used to control the rapid conversion of the laser beam between two different polarization states of horizontal and vertical polarized light. The phase modulation module performs phase modulation on the linearly polarized light to form saturated solid 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 the saturated solid and hollow light spots. The fluorescence detection module comprises a photomultiplier tube for collecting the saturated fluorescence signal and a lock-in amplifier for realizing correlation detection, amplification, filtering and demodulation of the fluorescence signal, the lock-in amplifier being controlled by a computer. The reference signal of the lock-in amplifier and the control signal of the electro-optical modulator are synchronized, the scanning signals of the saturated solid and hollow light spots varying with the adjustment of the polarization state of the excitation light by the electro-optical modulator are obtained by demodulation, and the microscopic imaging results are obtained by post-processing.

2. The nonlinear focal point modulation microscopic imaging device according to claim 1, wherein The electro-optical modulation module comprises an electro-optical modulator for modulating the polarization state of the excitation light and a high-voltage driving unit for controlling the electro-optical modulator, the high-voltage driving unit being controlled by a computer.

3. The nonlinear focal point modulation microscopic imaging device according to claim 1, wherein A polarizer is arranged 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 horizontal polarized light.

4. The nonlinear focal point modulation microscopic imaging device according to claim 3, wherein The excitation light passes through an electro-optical modulator, and the polarization state of the excitation light is not changed when no control voltage is applied to the electro-optical modulator, and the excitation light is converted into vertically polarized light when a voltage of a size of a half-wave voltage of the electro-optical modulator is applied to the electro-optical modulator. the electro-optical modulator is applied to the electro-optical modulator.

5. The nonlinear focal point modulation microscopic imaging device according to claim 1, wherein 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 a 0 to 2 pi vortex phase modulation pattern loaded by the phase modulation module when the excitation light reaches the surface of the spatial light modulator for the first time, 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 reaches the surface for the first time.

6. A method of nonlinear focal modulation microscopy for performing the nonlinear focal modulation microscopy apparatus of any one of claims 1-5, wherein, The method comprises the following steps: S1. The laser emits a laser beam with a power sufficient to cause a nonlinear saturation effect in the response of the fluorescent molecules, and a saturated fluorescence signal is obtained. S2. The laser beam is collimated and then passes through the electro-optical modulation module to control the rapid conversion of the excitation light between horizontal and vertical polarized light, and saturated solid and hollow light spots are obtained. S3. The saturated fluorescence signal is amplified, filtered and demodulated by the fluorescence detection module to obtain scanning images of the saturated solid and hollow light spots, and the scanning images are subjected to multi-image deconvolution processing to obtain the microscopic imaging results.

Images