A single optical path modulated fluorescence differential microscopy imaging device and method based on correlation detection technology

Abstract

The present invention discloses a single-light-path modulated fluorescence differential microscopy imaging device and method based on correlation detection technology, which relates to the field of confocal microscopy imaging technology. It includes: an illumination system for generating an excitation beam, a detection system for collecting the fluorescence signal emitted by the sample, and a computer for control and signal processing, and the illumination system, the detection system and the computer are connected in sequence. The present invention utilizes electro-optical modulation technology to achieve rapid conversion of the polarization state of the excitation light, enabling the laser to rapidly convert between solid and hollow states during the scanning process, effectively reducing the noise that may be brought by sample movement and system instability and the artifacts that may be introduced after differential operation.

Description

Technical Field

[0001] The present invention relates to the field of confocal microscopy imaging technology, and more specifically, to a single-path modulation fluorescence differential microscopy imaging device and method based on correlation detection technology. Background Art

[0002] Due to the limitation of the diffraction limit, the resolution that can be achieved by traditional far-field fluorescence microscopy technology is usually difficult to be lower than half wavelength, so its application in the fields of biomedicine, nanotechnology, and materials is greatly limited. In order to break through this limitation, since the 1990s, relevant researchers have proposed many super-resolution microscopy technologies such as stimulated emission depletion super-resolution microscopy (STED) and single molecule localization super-resolution microscopy (SMS). Among these super-resolution microscopy technologies, fluorescence differential microscopy (FED), as a recently proposed super-resolution microscopy technology, is based on confocal microscopy imaging technology. Two specific excitation spots are used to scan the sample to be measured to obtain two images, and the differential operation is performed on the two obtained images to improve the image resolution. Experiments have proved that the fluorescence differential microscopy imaging technology can achieve a resolution less than one-fourth wavelength in the far field and has a high signal-to-noise ratio. In addition, since high-power illumination light is not required, it can greatly reduce the possible photobleaching and photodamage and reduce the possible damage to the sample.

[0003] The traditional fluorescence differential microscopy imaging technology has problems that the imaging speed is limited and it cannot image samples with high requirements for time resolution, 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 between the solid and hollow spots, it is possible to perform the conversion between the solid and hollow spots during the scanning process, thereby improving the time resolution and reducing the phenomenon of artifacts that may occur due to sample movement. However, when performing high-speed modulation, a suitable detection method needs to be adopted to be able to timely acquire and extract the solid and hollow signals. The traditional fluorescence differential microscopy imaging technology usually uses an avalanche photodiode or a photomultiplier tube as a detector. Although this instrument can detect weak signals as a detector, it also amplifies the noise, and the imaging result often has certain noise. Especially when detecting weak signals, the influence of noise is more obvious, seriously affecting the imaging quality. The key to further improving the imaging quality is to find a way to reduce the collected noise.

[0004] Therefore, it is an urgent problem for those skilled in the art to propose a single-path modulation fluorescence differential microscopy imaging device and method based on correlation detection technology to solve the difficulties existing in the prior art. Summary of the Invention

[0005] In view of this, the present invention provides a single optical path modulation fluorescence differential microscopy imaging device and method based on correlation detection technology. The polarization modulation method is used to obtain solid and hollow light spots, and a lock-in amplifier that synchronizes the reference signal and the excitation light modulation signal is used as a detector to obtain fluorescence signals. Without introducing a second modulation optical path, it avoids errors and system instability that may be caused by optical path alignment and adjustment. The correlation detection method is used to demodulate the signals, effectively filtering out noise while obtaining the scanning results of solid and hollow light spots, and improving the imaging quality.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A single optical path modulation fluorescence differential microscopy imaging device based on correlation detection technology, comprising: an illumination system for generating an excitation beam, a detection system for collecting fluorescence signals emitted by a sample, and a computer for control and signal processing. The illumination system, the detection system and the computer are connected in sequence.

[0008] Optionally, the illumination system includes a laser, an electro-optic modulation module and a phase modulation module connected in sequence; wherein,

[0009] The laser is used to emit a laser beam;

[0010] The electro-optic modulation module is used to control the rapid conversion of the laser beam between two different polarization states of horizontal polarized light and vertical polarized light;

[0011] The phase modulation module is used to perform phase modulation on linearly polarized light to form solid and hollow light spots for illuminating the sample and exciting fluorescence.

[0012] Optionally, the electro-optic modulation module includes: an electro-optic modulator for modulating the polarization state of the excitation light and a high-voltage driving module for controlling the electro-optic modulator. The high-voltage driving module is controlled by the computer.

[0013] Optionally, a polarizer for adjusting the excitation light to an angle of 45° with the crystal optical axis direction of the electro-optic modulator is provided between the laser and the electro-optic modulation module. The adjusted excitation light is horizontal polarized light.

[0014] Optionally, the excitation light passes through the electro-optic modulator. When no control voltage is applied to the electro-optic modulator, the polarization state of the excitation light does not change. When a voltage of half-wave voltage V π of the electro-optic modulator is applied, the excitation light is converted into vertical polarized light. By applying a high-frequency pulse voltage of size V π with a fixed time interval to the electro-optic modulator, the rapid conversion of the excitation light between horizontal polarized light and vertical polarized light is achieved.

[0015] Optionally, the phase modulation module is a polarization-sensitive spatial light modulator. When the excitation light is horizontally polarized light, the spatial light modulator loaded with a vortex phase modulation pattern from 0 to 2π modulates the phase of the excitation light to obtain a hollow light spot. When the excitation light is vertically polarized light, the spatial light modulator does not modulate the phase of the excitation light to obtain a solid light spot.

[0016] Optionally, the detection system is used to collect, amplify, filter, and demodulate the fluorescence signal to obtain the scanning results of the solid light spot and the hollow light spot. The detection system includes a detector photomultiplier tube and a lock-in amplifier connected in sequence; where

[0017] The detector photomultiplier tube collects the fluorescence signal and transmits it to the lock-in amplifier;

[0018] The lock-in amplifier amplifies, filters, and demodulates the fluorescence signal.

[0019] Optionally, the reference signal of the lock-in amplifier is synchronized with the control signal of the electro-optic modulator and obtained through demodulation, and the scanning signals of the solid and hollow light spots that change as the electro-optic modulator adjusts the polarization state of the excitation light are obtained.

[0020] A single-path modulation fluorescence differential microscopy imaging method based on correlation detection technology, applying a single-path modulation fluorescence differential microscopy imaging device according to any one of the above, includes the following steps:

[0021] The laser beam emitted by the laser is adjusted to horizontally polarized light after collimation;

[0022] The electro-optic modulation module is used to control the rapid conversion of the excitation light between horizontally polarized light and vertically polarized light;

[0023] Phase modulation is performed on the linearly polarized light to form solid and hollow light spots for illuminating the sample and exciting fluorescence;

[0024] The detection system performs correlation detection, amplification, filtering, and demodulation on the fluorescence signal to obtain the scanning images of the solid and hollow light spots, and the microscopic imaging result is obtained after differential processing.

[0025] From the above technical solutions, it can be seen that compared with the prior art, the present invention discloses a single-path modulation fluorescence differential microscopy imaging device and method based on correlation detection technology, and its beneficial effects are:

[0026] (1) Based on the traditional fluorescence differential microscopy imaging system, a simple improvement is made. The system is relatively simple and applicable to all fluorescence differential microscopy imaging systems;

[0027] (2) Without introducing a second modulation optical path, the modulation of the excitation light is realized by a single optical path, avoiding the possible errors introduced when adjusting the coincidence of the optical paths.

[0028] (3) A method of adjusting the excitation light into solid and hollow light spots by using electro-optical modulation technology combined with a polarization-sensitive spatial light modulator for phase modulation is easier to implement compared with phase modulation, simplifying the optical system;

[0029] (4) Utilize electro-optical modulation technology to achieve rapid conversion of the polarization state of the excitation light, and achieve rapid conversion of the laser between solid and hollow states during the scanning process, effectively reducing the noise possibly brought by sample movement and system instability and the artifacts possibly introduced after differential operation;

[0030] (5) Use a lock-in amplifier to perform correlation detection on 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

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0032] Figure 1 It is a structural diagram of a single-path modulation fluorescence differential microscopy imaging device based on correlation detection technology provided by the present invention;

[0033] Figure 2 It is a flowchart of a single-path modulation fluorescence differential microscopy imaging method based on correlation detection technology provided by the present invention;

[0034] Figure 3 It is a schematic diagram of a traditional fluorescence differential microscopy imaging system device based on a confocal imaging system and galvanometer scanning provided by the present invention;

[0035] Figure 4 It is an example diagram of a single-path modulation fluorescence differential microscopy imaging system device based on correlation detection technology provided by the present invention;

[0036] Figure 5 It is a schematic diagram of using an electro-optical modulator to perform polarization modulation on the input light provided by the present invention;

[0037] Figure 6Schematic diagram of the spot intensity distribution obtained after modulation by a polarization-sensitive spatial light modulator and convergence by an optical system when the input light is vertically polarized light perpendicular to the modulation direction of the polarization-sensitive spatial light modulator or horizontally polarized light parallel to the modulation direction of the polarization-sensitive spatial light modulator; where 6a is the schematic diagram of the spot distribution in the horizontal plane of the vertically polarized light; 6b is the schematic diagram of the spot distribution in the longitudinal plane of the vertically polarized light; 6c is the schematic diagram of the spot distribution in the horizontal plane of the horizontally polarized light; 6d is the schematic diagram of the spot distribution in the longitudinal plane of the horizontally polarized light;

[0038] Figure 7 Schematic diagram of the process of obtaining a smaller solid point spread function by subtracting the point spread function obtained by illuminating with a hollow spot from the point spread function obtained by illuminating with a solid spot in optical differential microscopy provided by the present invention; where 7a is the schematic diagram of the point spread function obtained by illuminating with a solid spot; 7b is the schematic diagram of the point spread function obtained by illuminating with a hollow spot; 7c is the schematic diagram of the point spread function with a smaller full width at half maximum after subtraction;

[0039] Figure 8 Schematic diagram of the process of amplifying, demodulating, and filtering a signal using a lock-in amplifier in correlation detection provided by the present invention; where 8a is the distribution of the signal to be detected without introducing noise; 8b is the signal distribution after introducing noise; 8c is the distribution of the reference signal with the same frequency and phase as the signal to be detected; 8d is the signal distribution obtained after performing correlation detection on the noise signal using a lock-in amplifier;

[0040] Where, 1 - laser, 2 - single-mode fiber, 3 - collimating lens, 4 - polarizer, 5 - first mirror, 6 - spatial light modulator, 7 - second mirror, 8 - third mirror, 9 - 1 / 2 wave plate, 10 - 1 / 4 wave plate, 11 - dichroic mirror, 12 - galvanometer scanning system, 13 - scanning lens, 14 - field lens, 15 - fourth mirror, 16 - objective lens with a high numerical aperture, 17 - sample, 18 - filter, 19 - lens, 20 - multi-mode fiber, 21 - photomultiplier detector, 22 - computer, 23 - lock-in amplifier, 24 - electro-optic modulator, 25 - high-voltage drive module. Detailed implementation manners

[0041] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] See Figure 1As shown in the figure, the present invention discloses a single optical path modulated fluorescence differential microscopy imaging device based on correlation detection technology, including: an illumination system for generating an excitation beam, a detection system for collecting fluorescence signals emitted by the sample, and a computer 22 for control and signal processing. The illumination system, the detection system, and the computer 22 are connected in sequence.

[0043] Furthermore, the illumination system includes a laser 1, an electro-optic modulation module, and a phase modulation module connected in sequence; where

[0044] The laser 1 is used to emit a laser beam;

[0045] The electro-optic modulation module is used to control the rapid conversion of the laser beam between two different polarization states of horizontal polarized light and vertical polarized light;

[0046] The phase modulation module is used to perform phase modulation on linearly polarized light to form a solid light spot and a hollow light spot for illuminating the sample and exciting fluorescence.

[0047] Furthermore, the electro-optic modulation module includes: an electro-optic modulator 24 for modulating the polarization state of the excitation light and a high-voltage drive module 25 for controlling the electro-optic modulator 24. The high-voltage drive module 25 is controlled by the computer 22.

[0048] Specifically, the working principle of the electro-optic modulator 24 is the electro-optic effect, that is, the phenomenon that the refractive index of an electro-optic material changes under the action of a direct current or alternating current electric field. For an anisotropic electro-optic material, the electric field will affect the refractive index distribution of the material in each direction. The Pockels effect refers to the effect that the change in refractive index is proportional to the applied electric field, and 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 amplitude E of the external electric field and changes slightly with the change of E, and can be written as: Any polarized light can be decomposed into two orthogonal polarization components. In an anisotropic medium, these two polarization components are generally decomposed onto the 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, then their propagation speeds are c0 / n1 and c0 / n2 respectively, where c0 is the light wave speed in vacuum. Due to the difference in the propagation speeds of the two components, when one component propagates to the other end of the device, it will be ahead or behind the other polarization component in time, thus constituting a phase delay and changing the polarization state of the outgoing light. When an external electric field E is applied to an anisotropic material, for the Pockels effect, when light passes through a modulator with a length of L, the phase difference generated by the two polarization components is: For a transverse modulator with the direction of the applied electric field perpendicular to the direction of light propagation, its 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 inherent in the device, and the half-wave voltage is The phase delay is linearly related to the applied voltage. When the voltage is V π the phase delay is π.

[0049] Furthermore, a polarizer 4 is provided between the laser 1 and the electro-optic modulation module to adjust the excitation light to an angle of 45° with the crystal optical axis direction of the electro-optic modulator 24. The adjusted excitation light is horizontally polarized light.

[0050] Specifically, in the present invention, the incident laser is adjusted to linearly polarized light with a polarization direction at an angle of 45° with the crystal optical axis direction of the electro-optic modulator 24. In this case, the laser can be regarded as the sum of two components with equal amplitudes and orthogonal polarization directions parallel and perpendicular to the optical axis. When a voltage is applied to modulate the light beam by the electro-optic modulator 24, 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 magnitude is V π the phase difference between the two components changes by 180°, switching between two states where the phases are the same or differ by 180°, manifested as linearly polarized light switching between two states with perpendicular polarization directions.

[0051] Furthermore, the excitation light passes through the electro-optic modulator 24. When no control voltage is applied to the electro-optic modulator 24, the polarization state of the excitation light does not change. When a voltage with a magnitude equal to the half-wave voltage V π of the electro-optic modulator 24 is applied, the excitation light is converted into vertically polarized light. By applying a high-frequency pulsed voltage with a magnitude of V π having a fixed time interval to the electro-optic modulator 24, rapid switching of the excitation light between horizontally polarized light and vertically polarized light is achieved.

[0052] Furthermore, the phase modulation module is a polarization-sensitive spatial light modulator 6. When the excitation light is horizontally polarized light, the spatial light modulator 6 loaded with a vortex phase modulation pattern from 0 to 2π modulates the phase of the excitation light to obtain a hollow light spot. When the excitation light is vertically polarized light, the spatial light modulator 6 does not modulate the phase of the excitation light, obtaining a solid light spot.

[0053] The principle of the present invention is as follows:

[0054] Based on the traditional fluorescence differential microscopy imaging system, the present invention uses polarization modulation combined with phase modulation as the method for obtaining solid and hollow light spots. After collimation and beam expansion, the laser first passes through a polarizer 4 with the polarization direction at an angle of 45° to the crystal optical axis direction of the electro-optic modulator 24 to be adjusted to horizontally polarized light, and then enters the electro-optic modulator 24. Under the control of the electro-optic modulator 24, the laser realizes a rapid conversion between horizontal and vertical polarized lights with the polarization direction at an angle of ±45° to the crystal optical axis direction of the electro-optic modulator 24. After the phase modulation by the polarization-sensitive spatial light modulator 6 loaded with a phase modulation pattern of 0 - 2π, the horizontally polarized light is modulated by the spatial light modulator 6 and converges into a hollow light spot after passing through the optical microscopy imaging system, while the vertically polarized light is not modulated by the spatial light modulator 6 and converges into a solid light spot after passing through the optical microscopy imaging system. The sample 17 is scanned with the solid and hollow light spots respectively, and the obtained imaging results are subtracted according to a certain ratio to obtain the fluorescence differential microscopy imaging result.

[0055] In the microscopy imaging system, the light spot intensity distribution near the focus 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 using a high numerical aperture objective 16, according to the vector diffraction theory, the electric field vector distribution near the focus can be expressed as:

[0056]

[0057] Where, is the electric field vector at the point (r2, φ2, z2), (r2, φ2, z2) is the cylindrical coordinate distribution with the origin at the focus, C is the normalized constant, A1(θ, φ) is the amplitude function of the input light, usually expressed as 1 or a deformation of the basic Gaussian function, A2(θ, φ) is a 3×3 matrix representing the imaging lens structure, usually expressed as:

[0058]

[0059] , [p x ; p y ; p z is the unit matrix vector representing the polarization state of the input light.

[0060] In the fluorescence differential microscopy imaging system, by using a phase plate or the spatial light modulator 6, a phase delay can be introduced 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:

[0061]

[0062] Among them, Δα(θ, φ) is the introduced phase delay. Usually, a vortex phase plate from 0 to 2π is used to generate a hollow light spot, and the introduced phase delay is expressed as Δα = φ. By substituting the above formula, it can be obtained that when the input light is horizontally polarized light, the distribution of the light spot obtained at the focal point after the phase modulation of the spatial light modulator 6 in the horizontal plane is a hollow light spot. When the input light is vertically polarized light, the distribution of the light spot obtained at the focal point without the phase modulation of the spatial light modulator 6 in the horizontal plane is a solid light spot.

[0063] Furthermore, the detection system is used to collect, amplify, filter, and demodulate the fluorescence signal to obtain the scanning results of the solid light spot and the hollow light spot. The detection system includes a detector photomultiplier tube 21 and a lock-in amplifier 23 connected in sequence; among them,

[0064] The detector photomultiplier tube 21 collects the fluorescence signal and transmits it to the lock-in amplifier 23;

[0065] The lock-in amplifier 23 amplifies, filters, and demodulates the fluorescence signal.

[0066] Furthermore, the reference signal of the lock-in amplifier 23 is synchronized with the control signal of the electro-optic modulator 24 and obtained through demodulation, and the scanning signals of the solid and hollow light spots that change as the electro-optic modulator 24 adjusts the polarization state of the excitation light are obtained.

[0067] Specifically, the lock-in amplifier 23 is a weak signal correlation detection device based on the cross-correlation reception theory, which can effectively suppress noise using a phase-sensitive detector and is usually composed of three main parts: a signal channel, a reference channel, and a phase-sensitive detection. Among them, the signal channel amplifies and narrow-band filters the detected signal and then outputs the signal U s , and the reference channel provides a square wave signal U r that is in the same frequency as the measured signal and has an adjustable phase through a trigger circuit, a frequency multiplier circuit, a phase shift circuit, and a square wave drive circuit. s The phase-sensitive detection consists of a multiplier, a low-pass filter, and a DC amplifier. The input signal U r and the reference signal U s are mixed in the phase-sensitive detector, and after passing through the low-pass filter, a DC output component proportional to the amplitude of the input signal is obtained. When the input signal U r and the reference signal U s are sine waves: U sm = U r cosωt, U rm = U s cos(ωt + φ). After mixing through the multiplier, the output signal is: where φ is the phase difference between the input signal U rAfter the input signal and the reference signal are correlated, the spectrum of the output signal is transformed from ω to the frequency band of the difference frequency 0 and the sum frequency 2ω. 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 lock-in amplifier 23 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. The noise output is an AC signal and can be filtered out by the subsequent low-pass filter. Therefore, the lock-in amplifier 23 can extract the effective signal from the noise. If the low-pass filter in the phase-sensitive detector is a first-order RC filter, the transfer function is: The corresponding equivalent noise bandwidth is: Since the measured signal and the reference signal are synchronized in the lock-in amplifier 23, there is no frequency stability issue. Therefore, it can be regarded as a tracking filter with a high-quality Q and 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 lock-in amplifier 23 is expressed as: Among them, SNR o is the output signal-to-noise ratio of the lock-in amplifier 23, SNR i is the input signal-to-noise ratio of the lock-in amplifier 23, Δf eo is the output noise bandwidth, Δf ei is the noise bandwidth of the input.

[0068] Combining electro-optical modulation technology with detection by a lock-in amplifier 23, the electro-optical modulator 24 is used to rapidly convert the input light between horizontally and vertically polarized light. The resulting light spot, modulated by the polarization-sensitive electro-optical modulator 24 and then fed into the optical microscopy imaging system, rapidly switches between solid and hollow. Rapid conversion between solid and hollow light is achieved during the scanning process. A signal with the same frequency and phase as the drive signal of the electro-optical modulator 24 is used as the reference signal for the lock-in amplifier 23. After demodulation, filtering, and amplification, detected noise and interference signals are effectively filtered out, extracting only the signal with the same frequency and phase as the reference signal, significantly improving the final imaging quality and signal-to-noise ratio.

[0069] and Figure 1 Corresponding to the device described above, the present invention also provides a single light path modulation fluorescence differential microscopy imaging method based on correlation detection technology for Figure 1 For the specific implementation of the device, see the flowchart of the method. Figure 2 As shown, the following steps are included:

[0070] The laser beam emitted by the laser 1 is adjusted to horizontal polarized light after collimation;

[0071] The electro-optic modulation module is used to control the rapid conversion of the excitation light between horizontal polarized light and vertical polarized light;

[0072] Phase modulation is performed on the linearly polarized light to form solid spots and hollow spots for illuminating the sample and exciting fluorescence;

[0073] The fluorescence signal is subjected to correlation detection, amplification, filtering, and demodulation by the detection system to obtain the scanned images of the solid spots and hollow spots, and the microscopic imaging result is obtained after differential processing.

[0074] Specifically, refer to Figure 3 The figure shows a schematic diagram of a traditional fluorescence differential microscopy imaging system based on a confocal imaging system and galvanometer scanning. Figure 4 The figure is an example diagram of a single-path modulation fluorescence differential microscopy imaging system based on correlation detection technology, including: laser 1, single-mode fiber 2, collimating lens 3, polarizer 4, first mirror 5, spatial light modulator 6, second mirror 7, third mirror 8, 1 / 2 wave plate 9, 1 / 4 wave plate 10, dichroic mirror 11, galvanometer scanning system 12, scanning lens 13, field lens 14, fourth mirror 15, high numerical aperture objective lens 16, sample 17, filter 18, lens 19, multimode fiber 20, detector photomultiplier tube 21, computer 22, lock-in amplifier 23, electro-optic modulator 24, high voltage drive module 25.

[0075] Among them, 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 direction of the electro-optical modulator 24 and parallel to the modulation direction of the polarization-sensitive spatial light modulator 6, so as to facilitate the polarization modulation of the electro-optical modulator 24 and the phase modulation of the spatial light modulator 6; the electro-optical modulator 24 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 direction of the electro-optical modulator 24, that is, horizontal and vertical linear polarized light; the polarization-sensitive spatial light modulator 6 is used to modulate the linear polarized light into a hollow light spot or a solid light spot by loading a vortex phase modulation pattern of 0 to 2π; the second reflector 7 and the third reflector 8 are used to compress the optical path; the 1 / 2 wave plate 9 and The 1 / 4 wave plate 10 is used to adjust the polarization state of the modulated excitation light to circularly polarized light; the scanning lens 13 and the field lens 14 form a 4f system, which is used to match the beam size of the excitation light with the numerical aperture of the high numerical aperture objective lens 16, and ensure that the entrance pupil position of the high numerical aperture objective lens 16 is parallel light; the light band of the filter 18 needs to match the fluorescence band of the sample 17; the reference signal of the phase-locked amplifier 23 needs to be at the same frequency and phase as the driving signal of the electro-optical modulator 24; the drive of the galvanometer scanning system 12 needs to match the driving signal of the electro-optical modulator 24. For each imaging unit, after obtaining the imaging results under solid and hollow illumination conditions, the galvanometer scanning system 12 is controlled to scan and image the next imaging unit.

[0076] use Figure 4 The process of implementing fluorescence differential microscopy imaging with the device shown is as follows:

[0077] (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 after passing through the polarizer 4, with the polarization direction being at an angle of 45° to the crystal optical axis of the electro-optical modulator 24 and parallel to the modulation direction of the polarization-sensitive spatial light modulator 6;

[0078] (2) Horizontally polarized light enters the electro-optic modulator 24 for polarization modulation. Under the control of the high voltage driving module 25, 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 direction of the electro-optic modulator 24. When the driving voltage is the half-wave voltage V π When the excitation light is large or small, it is converted into vertical linear polarized light with a polarization direction of -45° to the crystal optical axis direction of the electro-optical modulator 24. As the driving voltage changes, the excitation light quickly switches between the two polarization directions of linear polarized light. The electro-optical modulator 24 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;

[0079] (3) The linearly polarized light is reflected by the first mirror 5 and then enters the polarization-sensitive spatial light modulator 6 controlled by the computer 22 and loaded with a vortex phase modulation pattern from 0 to 2π for phase modulation. When the linearly polarized light is horizontally polarized light, the spatial light modulator 6 modulates the laser to obtain a hollow spot. When the linearly polarized light is vertically polarized light, the spatial light modulator 6 does not modulate the laser to obtain a solid spot; the excited light after being phase-modulated passes through the second mirror 7 and the third mirror 8, and then passes through the half-wave plate 9 and the quarter-wave plate 10 to compensate for the phase differences caused by the second mirror 7, the third mirror 8, and the subsequent dichroic mirror 11, galvanometer scanning system 12 and the fourth mirror 15, and is adjusted to make the modulated excited light circularly polarized; the excited light enters the galvanometer scanning system 12 after being reflected by the dichroic mirror 11 and realizes two-dimensional scanning on the surface of the final sample 17; the excited light coming out of the galvanometer scanning system 12 passes through the 4f system composed of the scanning lens 13 and the field lens 14, and the beam size is adjusted to match the numerical aperture of the high-numerical-aperture objective lens 16; the excited light is focused by the high-numerical-aperture objective lens 16 to obtain an excitation spot limited by the diffraction limit, and the excitation spot is focused on the fluorescent sample 17;

[0080] (4) The excited light that rapidly switches between the solid and hollow spot modes illuminates the fluorescent sample 17 and excites the sample 17 to generate fluorescence. The generated fluorescence is collected by the high-numerical-aperture objective lens 16, and then passes through the fourth mirror 15, field lens 14, scanning lens 13, and galvanometer scanning system 12 and is incident on the dichroic mirror 11; the dichroic mirror 11 transmits the fluorescence; the transmitted fluorescence passes through the filter 18 to filter out the excited light reflected by the sample 17 and other stray light, and only allows the fluorescence to exit; the exiting fluorescence is converged by the lens 19 and focused on the multimode optical fiber 20, and is transmitted by the multimode optical fiber 20 to the detector photomultiplier tube 21; the detector photomultiplier tube 21 converts the optical signal into an electrical signal and transmits the electrical signal to the lock-in amplifier 23;

[0081] (5) The computer 22 controls the electro-optic modulator 24 through the high-voltage driving module 25 to realize the rapid switching of the excited light between the solid and hollow illumination modes. The computer 22 controls the reference signal of the lock-in amplifier 23 to be consistent with the control signal of the high-voltage driving module 25 of the electro-optic modulator 24. The lock-in amplifier 23 performs correlation detection on the signal detected by the detector photomultiplier tube 21 to obtain two imaging results corresponding to each scanning imaging unit in the solid or hollow illumination mode. The computer 22 subtracts the two imaging results according to a certain ratio to obtain the final image corresponding to one scanning imaging unit;

[0082] (6) The galvanometer scanning system 12 is connected to the computer 22, and the computer 22 controls the galvanometer scanning system 12 to perform two-dimensional scanning on the sample 17 to obtain the two-dimensional image corresponding to the sample 17.

[0083] As shown Figure 5 in FIG. 2, the change in the voltage applied to the electro-optic modulator 24 introduces a variable phase delay between the two polarization states of the input light. For horizontally polarized light with a polarization direction at an angle of 45° to the crystal optical axis of the electro-optic modulator 24, when the magnitude of the driving voltage of the electro-optic modulator 24 is the half-wave voltage V π of the electro-optic modulator 24, a phase delay of π is introduced between the two polarization states of the input light, and the polarization direction of the linearly polarized light is changed to vertically polarized light with an angle of -45° to the crystal optical axis of the electro-optic modulator 24. When the magnitude of 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° to the crystal optical axis of the electro-optic modulator 24. By applying a high-frequency pulse voltage with a magnitude of V π at intervals, rapid conversion of the light beam between two states of horizontally and vertically polarized light with polarization directions at angles of 45° and -45° to the crystal optical axis of the electro-optic modulator 24 can be achieved.

[0084] Referring to Figure 6 FIG. 3, when the input light is vertically polarized light with a polarization direction perpendicular to the modulation direction of the polarization-sensitive spatial light modulator 6, the spatial light modulator 6 does not modulate the vertically polarized light, and a solid light spot is obtained after being converged by the optical system. According to the vector diffraction theory, the schematic diagram of the light spot intensity distribution obtained by the solid light spot after being converged by the optical system is calculated. The schematic diagram of the horizontal plane light spot distribution of the vertically polarized light is shown in FIG. 6a, and the schematic diagram of the longitudinal plane light spot distribution of the vertically polarized light is shown in FIG. 6b. When the input light is horizontally polarized light with a polarization direction parallel to the modulation direction of the polarization-sensitive spatial light modulator 6, the spatial light modulator 6 loaded with a vortex phase modulation pattern from 0 to 2π modulates the horizontally polarized light, and a hollow light spot is obtained after being converged by the optical system. According to the vector diffraction theory, the specific distribution of the hollow light spot near the focus of the optical system is calculated. The schematic diagram of the horizontal plane light spot distribution of the horizontally polarized light is shown in FIG. 6c, and the schematic diagram of the longitudinal plane light spot distribution of the horizontally polarized light is shown in FIG. 6d.

[0085] Referring to Figure 7As shown in the figure, it is a schematic diagram of the process of obtaining a smaller solid point spread function by subtracting the point spread function obtained by illuminating with a solid light spot from the point spread function obtained by illuminating with a hollow light spot. The vertical linearly polarized light that is not modulated by the spatial light modulator 6 passes through the optical system and converges to illuminate the fluorescent sample 17 with a solid light spot, and then excites the sample 17 to generate fluorescence of the solid point spread function. That is, the schematic diagram of the point spread function obtained by illuminating with a solid light spot is shown in Fig. 7a. The horizontal linearly polarized light modulated by the spatial light modulator 6 passes through the optical system and converges to illuminate the fluorescent sample 17 with a hollow light spot, and then excites the sample 17 to generate fluorescence of the hollow point spread function. That is, the schematic diagram of the point spread function obtained by illuminating with a hollow light spot is shown in Fig. 7b. After subtracting the two fluorescence signals, the final imaging result is obtained. The solid point spread function obtained by subtracting the hollow point spread function has a smaller full width at half maximum. That is, the schematic diagram of the point spread function with a smaller full width at half maximum after subtraction is shown in Fig. 7c.

[0086] See Figure 8 As shown in the figure, it is the result of performing correlation detection on a signal with certain noise by a lock-in amplifier 23 using a reference signal with the same frequency and phase as the signal to be detected to filter out the noise. The distribution diagram of the signal to be detected without introducing noise is shown in Fig. 8a. The distribution of the signal to be detected with a certain Gaussian noise introduced over time, that is, the distribution diagram of the signal after introducing noise is shown in Fig. 8b. The signal containing noise detected by an ordinary detector is shown in Fig. 8b. When the reference signal of the lock-in amplifier 23 is a cosine signal with the same frequency and phase as the signal to be detected, that is, the distribution of the reference signal with the same frequency and phase as the signal to be detected is shown in Fig. 8c. The detection signal result that is basically consistent with the frequency and phase distribution of the signal to be detected after filtering out the noise obtained by the lock-in amplifier 23 performing correlation detection. That is, the signal distribution obtained by performing correlation detection on the noise signal by the lock-in amplifier 23 is shown in Fig. 8d.

[0087] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same and similar parts among the embodiments, reference can be made to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the description of the method part.

[0088] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can 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 these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A single-light-path modulation fluorescence differential microscopy imaging device based on correlation detection technology, characterized in that: include: An illumination system for generating an excitation beam, a detection system for collecting fluorescence signals emitted by the sample, and a computer for control and signal processing, wherein the illumination system, the detection system, and the computer are connected in sequence; The lighting system includes a laser, an electro-optical modulation module and a phase modulation module connected in sequence; wherein, a laser for emitting a laser beam; Electro-optical modulation module, used to control the rapid conversion of the laser beam between two different polarization states: horizontal polarization and vertical polarization; A phase modulation module is used to phase modulate linearly polarized light to form solid and hollow light spots that illuminate the sample and excite fluorescence; The phase modulation module is a polarization-sensitive spatial light modulator. When the excitation light is horizontally polarized, the spatial light modulator loaded with a vortex phase modulation pattern from 0 to 2π phase modulates the excitation light to obtain a hollow light spot. When the excitation light is vertically polarized, the spatial light modulator does not phase modulate the excitation light to obtain a solid light spot. The electro-optical modulation module includes: an electro-optical modulator for modulating the polarization state of the excitation light and a high-voltage driving module for controlling the electro-optical modulator, wherein the high-voltage driving module is controlled by a computer; A polarizer 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 of the electro-optical modulator. The adjusted excitation light is horizontally polarized light.

2. The single-light-path modulation fluorescence differential microscopy imaging device based on correlation detection technology according to claim 1, 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 of the electro-optic modulator, the polarization state of the excitation light does not change. When the voltage of magnitude is 0.01, the excitation light is converted into vertically polarized light. The high-frequency pulse voltage is used to achieve rapid conversion of the excitation light between horizontally polarized light and vertically polarized light.

3. The single-light-path modulation fluorescence differential microscopy imaging device based on correlation detection technology according to claim 1, characterized in that: The detection system is used to collect, amplify, filter and demodulate the fluorescence signal to obtain the scanning results of solid and hollow spots. The detection system includes a detector, a photomultiplier tube and a lock-in amplifier connected in sequence; wherein, The detector photomultiplier tube collects the fluorescence signal and transmits it to the lock-in amplifier; Lock-in amplifier amplifies, filters and demodulates the fluorescence signal.

4. The single-light-path modulation fluorescence differential microscopy imaging device based on correlation detection technology according to claim 3, characterized in that: The reference signal of the lock-in amplifier is synchronized with the control signal of the electro-optical modulator, and the scanning signals of the solid and hollow light spots are obtained by demodulation, which change as the electro-optical modulator adjusts the polarization state of the excitation light.

5. A single-path modulated fluorescence differential microscopy imaging method based on correlation detection technology, characterized in that A single-light-path modulation fluorescence differential microscopy imaging device based on correlation detection technology according to any one of claims 1 to 4 comprises the following steps: The laser beam emitted by the laser is adjusted to horizontal polarized light after collimation; The electro-optical modulation module is used to control the rapid conversion of the excitation light between horizontal polarization light and vertical polarization light; Phase modulate the linearly polarized light to form solid and hollow spots that illuminate the sample and excite fluorescence; The fluorescence signal is correlated detected, amplified, filtered and demodulated by the detection system to obtain scanning images of solid and hollow spots, and the microscopic imaging results are obtained after differential processing.

Images