Differential nonlinear competitive super-resolution microscopy method and device

Abstract

The invention discloses a differential nonlinear competitive super-resolution microscopy method and a differential nonlinear competitive super-resolution microscopy device. Light beams emitted by the same light source simultaneously irradiate the surface of a fluorescent sample. Wherein one light beam is focused on a sample after passing through a spatial light modulator to form a high-energy hollow light spot for generating a saturation effect; the other beam of light is modulated into time sequence light by an acousto-optic modulator, the time sequence light is focused on the sample to form a low-energy solid light spot, and signal light emitted by each scanning point of the sample is collected; and performing intensity control on the light intensity of the hollow light through a light intensity modulation device to obtain two nonlinear competition result images, and performing differential processing to obtain a super-resolution image. The device is simple and convenient to operate; on the basis of a competition mechanism of solid spots and hollow spots during sample fluorescence excitation, a high-resolution image can be obtained under low illumination light intensity by utilizing differential processing, and negative values of the differentiated image are greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the field of super-resolution, and in particular relates to a super-resolution microscopy method and device for realizing a resolution beyond the diffraction limit in a far field by utilizing the principle of fluorescence saturation. Background Art

[0002] Due to the influence of diffraction in the optical system, the resolution that can be achieved by conventional far-field optical microscopy methods is limited. According to the Abbe diffraction limit theory, the size of the spot formed by the beam after being focused by the microscope objective is expressed in terms of the full width at half maximum: ,in is the working wavelength of the microscope, is the numerical aperture of the microscope objective used. Therefore, the limiting resolution of conventional far-field optical microscopes is generally limited to about half a wavelength.

[0003] In order to break through the limitation of optical diffraction limit and improve the resolution of microscopic system, researchers have proposed a variety of super-resolution optical microscopy methods, among which fluorescence excitation has received great attention. Some commonly used methods include: stimulated emission depletion microscopy (STED), structured illumination fluorescence microscopy (SIM), stochastic optical reconstruction microscopy (STORM), etc.

[0004] These methods can all achieve fluorescence super-resolution microscopy in the far field and have been applied accordingly in actual tests, but they all still have shortcomings. Among them, the resolution of STED microscopy is determined by the optical power of the added loss light. Therefore, when achieving high resolution, the required optical power is very strong, which can easily lead to bleaching of fluorescent molecules. Although SIM microscopy does not require high optical power, it requires raster scanning, has a slow imaging speed, and a more complex imaging system. The imaging speed of STORM microscopy is also very slow, and it is currently difficult to use it in real-time detection of living cells.

[0005] In conventional fluorescence microscopy, the fluorescence intensity emitted by the sample is proportional to the intensity of the excitation light; when the excitation light intensity continues to increase, the number of fluorescent molecules in the excited state will reach saturation, the proportional relationship will no longer hold, and the point spread function of the fluorescence will also change, which can be used to obtain more detailed structural information. Therefore, a super-resolution microscopy method based on saturated excitation, saturated absorption competition microscopy (SAC), was proposed and applied in the fields of biology and materials. Since this method requires a strong saturated hollow light intensity, a method to reduce the hollow light intensity is needed. Recently, a method called fluorescence differential microscopy (FED) has been proposed. Through differential processing, super-resolution images can be obtained at lower light intensities. However, differential processing will cause the final image to produce negative values ​​and introduce artifacts. Summary of the invention

[0006] The present invention provides a method and device for improving spatial resolution in super-resolution microscopy, which can obtain multiple nonlinear competitive imaging images by changing the hollow light intensity to achieve the purpose of reducing negative values. The method and device have the characteristics of simple device, low light damage, high resolution, etc., and can be well applied to the detection of fluorescent and non-fluorescent samples.

[0007] In a first aspect, the present invention provides a differential nonlinear competitive super-resolution microscopy method comprising the following steps:

[0008] Step 1) adjusting the polarization of the collimated laser beam so that the intensity of the S polarization component is much greater than the intensity of the P polarization component;

[0009] Step 2) dividing the laser beam processed in step 1) into two linearly polarized beams, namely, P light and S light;

[0010] Step 3) performing phase modulation on the S light;

[0011] Step 4) combining the phase-modulated S light and P light into one beam, and then converting it into left-handed circularly polarized light and right-handed circularly polarized light according to the polarization characteristics;

[0012] Step 5) projecting the light beam converted into left-handed circularly polarized light and right-handed circularly polarized light onto the sample to scan the sample;

[0013] Step 6) receiving the signal light emitted by the sample during scanning to obtain the first image;

[0014] Step 7) By changing the intensity of the S light, the second image is obtained;

[0015] Step 8) Perform differential processing on the results of step 6) and step 7), set the differential coefficient, return negative values ​​to zero, and finally obtain a super-resolution image.

[0016] In a second aspect, the present invention provides a differential nonlinear competitive super-resolution microscopy device, comprising:

[0017] It comprises a light source and a sample stage for carrying a sample to be tested, wherein the light source and the sample stage are provided in sequence:

[0018] A first 1 / 2 wave plate, used to change the polarization characteristics of the light beam emitted by the light source;

[0019] A polarization beam splitter, used to split the light beam after the polarization property is changed into two linearly polarized lights, wherein the two linearly polarized lights are P light and S light respectively;

[0020] A spatial light modulator, used for phase modulating the S light;

[0021] an acousto-optic tunable filter, used for controlling the intensity of the S light;

[0022] The second 1 / 2 wave plate and the first 1 / 4 wave plate are used to change the polarization characteristics of the polarized light;

[0023] A polarization beam combiner, used to combine the P light passing through the spatial light modulator and the S light passing through the acousto-optic modulator into one beam;

[0024] The second quarter wave plate is used to change the polarization characteristics of the light in the light beam emitted by the polarization beam combiner;

[0025] The sample scanning system is used to deflect the optical path of the light beams converted into left-handed circularly polarized light and right-handed circularly polarized light by the second quarter wave plate in sequence so as to project them onto the sample, and converge the phase-modulated P light into a hollow light spot on the sample, and converge the S light into a solid light spot on the sample to scan the sample;

[0026] The detection system is used to collect the signal light emitted by the sample.

[0027] Compared with the prior art, the present invention has the following beneficial technical effects:

[0028] (1) A saturation excitation competition microscopy method was proposed to improve resolution;

[0029] (2) Effectively alleviate the negative value phenomenon;

[0030] (3) The device is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a schematic structural diagram of the super-resolution microscopy device of this embodiment;

[0032] Figure 2 PSF comparison of the imaging in this embodiment;

[0033] Figure 3 is the cross-sectional intensity comparison of the imaging PSF of this embodiment;

[0034] Figure 4 This is the imaging comparison of nano fluorescent particles in this embodiment. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings, but the present invention is not limited thereto.

[0036] The present application embodiment provides a differential nonlinear competitive super-resolution microscopy method, comprising the following steps:

[0037] 1) Adjust the polarization of the collimated laser beam so that the intensity of the S polarization component is much greater than that of the P polarization component;

[0038] 2) Splitting the laser beam processed in step 1) into two linearly polarized beams, namely, P light and S light;

[0039] 3) performing phase modulation on the S light;

[0040] 4) Combine the phase-modulated S light and P light into one beam, and then convert it into left-handed circularly polarized light and right-handed circularly polarized light according to the polarization characteristics;

[0041] 5) Projecting the light beams converted into left-handed circularly polarized light and right-handed circularly polarized light onto the sample to scan the sample, so that the phase-modulated P light converges into a hollow light spot on the sample and the S light converges into a solid light spot on the sample during scanning;

[0042] 6) Receive the signal light emitted by the sample during scanning and obtain the first image;

[0043] 7) By changing the intensity of the S light, the second image is obtained;

[0044] 8) Perform differential processing on the results of 6) and 7), set the differential coefficient, return negative values ​​to zero, and finally obtain a super-resolution image.

[0045] Furthermore, when the sample to be tested (i.e., the sample) is a fluorescent sample, the signal light is the fluorescence excited on the sample by the circularly polarized light after being projected by the microscope objective lens;

[0046] Furthermore, when the sample to be tested is a non-fluorescent sample (such as a gold nanoparticle sample), the signal light is a scattered light beam that is projected by the microscope objective lens and then passes through the sample surface.

[0047] The x and y axis directions on the sample to be tested are determined by the two-dimensional scanning direction.

[0048] Preferably, in the super-resolution microscopy method of the present application, a spatial light modulator is used to perform phase modulation on the S light.

[0049] The present application also provides a differential nonlinear competitive super-resolution microscopy device, comprising a light source and a sample stage for carrying a sample to be tested, wherein the light source and the sample stage are provided in sequence with:

[0050] A first 1 / 2 wave plate for changing the polarization property of the light beam emitted by the light source;

[0051] A polarization beam splitter for splitting the light beam after the polarization property is changed into two linearly polarized light beams, wherein the two linearly polarized light beams are P light and S light respectively;

[0052] A spatial light modulator for phase modulating the S light;

[0053] A second 1 / 2 wave plate and a first 1 / 4 wave plate for changing the polarization characteristics of the polarized light;

[0054] A polarization beam combiner for combining the P light passing through the spatial light modulator and the S light passing through the acousto-optic modulator into one beam;

[0055] A second quarter wave plate for changing the polarization characteristics of light in the light beam emitted from the polarization beam combiner;

[0056] A scanning galvanometer system, a scanning lens, a field lens and a microscope objective lens are used to deflect the optical path of the light beams that are converted into left-handed circularly polarized light and right-handed circularly polarized light by the second 1 / 4 wave plate so as to project them onto the sample, and converge the phase-modulated P light into a hollow light spot on the sample, and converge the S light (sequential light) into a solid light spot on the sample to scan the sample, and the scanning galvanometer system, the scanning lens, the field lens and the microscope objective lens are arranged in sequence;

[0057] A detection system for collecting signal light emitted by the sample;

[0058] and a controller for controlling the spatial light modulator and the scanning galvanometer system.

[0059] Further, in the embodiment of the present application, the circularly polarized light is projected onto the sample to be tested through a microscope objective lens, as follows:

[0060] The scanning galvanometer system deflects the optical path of the light beam passing through the second quarter wave plate;

[0061] The scanning lens and the field lens are used to focus and collimate the light beam emitted by the scanning galvanometer system respectively.

[0062] Furthermore, in the embodiment of the present application, the signal light emitted by the sample is received by a detection system, and the detection system includes:

[0063] A beam splitter is arranged between the spatial light modulator and the scanning galvanometer system. When the sample to be measured is a fluorescent sample, a dichroic mirror should be used for the beam splitter.

[0064] A bandpass filter for filtering out stray light in the signal light emitted by the beam splitter, wherein the bandpass filter can be removed when the sample to be tested is a non-fluorescent sample, such as by using an electric device of a flip mirror;

[0065] A detector for detecting the light intensity signal of the signal light beam, wherein the detector is a photomultiplier tube (PMT);

[0066] a collecting lens for focusing the filtered signal beam onto a detector;

[0067] The spatial filter for spatially filtering the signal light beam is located at the focal plane of the collecting lens. The spatial filter can be a pinhole or a multimode optical fiber. If a pinhole is used, the diameter of the pinhole should be smaller than an Airy disk diameter.

[0068] Preferably, a single-mode optical fiber for filtering the laser beam and a collimating lens for collimating the laser beam are sequentially arranged between the light source and the 1 / 2 wave plate.

[0069] Example:

[0070] like Figure 1 As shown, the super-resolution microscopy device of this embodiment includes: a laser 1, a reflector 2, a single-mode optical fiber 3, a collimating lens 4, a 1 / 2 wave plate 5 (i.e., a first 1 / 2 wave plate), a polarization beam splitter 6, a delay device 7, an acousto-optic modulator 8, a reflector 9, a first 1 / 4 wave plate 10 (i.e., a first 1 / 4 wave plate), a 1 / 2 wave plate 11 (i.e., a second 1 / 2 wave plate), an acousto-optic tunable filter 12, a reflector 13, a spatial light modulator 14, a polarization beam combiner 15, a dichroic mirror 16, a reflector 17, a scanning galvanometer 18, a second 1 / 4 wave plate 19 (i.e., a second 1 / 4 wave plate), a reflector 20, a scanning lens 21, a field lens 22, a microscope objective 23, a sample stage 24, a bandpass filter 25, a collecting lens 26, a pinhole 27, a multimode optical fiber 28, a dielectric reflector 29 and a detector 30.

[0071] Among them, the reflector 2, the single-mode optical fiber 3, the collimating lens 4, the 1 / 2 wave plate 5, the polarization beam splitter 6, the delay device 7, the acousto-optic modulator 8, the reflector 9, the 1 / 4 wave plate 10, and the 1 / 2 wave plate 11 are sequentially located on the optical axis of the outgoing light beam of the laser 1; the transmission axis direction of the 1 / 2 wave plate 5 is adjusted so that the intensity of the S polarization component after the light beam passes through is much greater than the intensity of the P polarization component, with a difference of two orders of magnitude.

[0072] The acousto-optic modulator 8 is located on the optical axis of the light beam modulated by the polarization beam splitter 6 .

[0073] The time delay device 7 is used to adjust the optical path lengths of the P light and the S light so that the arrival times of the P light and the S light on the sample surface are equal.

[0074] The polarization beam combiner 15 is located on the optical axis of the light beam modulated by the spatial light modulator 14 .

[0075] The light beam will be diffracted after passing through the acousto-optic modulator 8, generating multiple orders of diffracted light. In this embodiment, only the first order of diffracted light is collected.

[0076] The dichroic mirror 16 and the reflector 17 are sequentially located on the optical axis of the outgoing light of the polarization beam combiner 15 after being deflected by the reflector; the scanning galvanometer 18 is located on the optical axis of the light beam after being reflected by the reflector 17 .

[0077] Among them, the quarter wave plate 19 (achromatic), the scanning lens 21, the field lens 22, the microscope objective lens 23, and the sample stage 24 are sequentially located on the optical axis of the outgoing light beam of the scanning galvanometer 18.

[0078] Among them, the bandpass filter 25, the collecting lens 26, the pinhole 27, the multimode optical fiber 28, the dielectric reflector 29, and the detector 30 are sequentially located on the optical axis after transmission through the dichroic mirror 16; the pinhole 27 is located at the focal plane of the collecting lens 26.

[0079] The controller is connected to the spatial light modulator 14 and is used to load the modulation phase of the spatial light modulator 14 .

[0080] In the above device, the numerical aperture NA of the microscope objective lens 23 is 1.4; the diameter of the pinhole 27 used is 0.43 Airy disk diameter, and the detector 30 is a photomultiplier tube (PMT).

[0081] use Figure 1 The method for performing super-resolution microscopy with the apparatus shown is as follows:

[0082] The laser beam emitted from the laser 1 is first introduced into the single-mode optical fiber 3, and the laser beam emitted from the single-mode optical fiber 3 is collimated by the collimating lens 4. The collimated beam is incident on the first 1 / 2 wave plate 5 to change the polarization, so that the intensity of the S polarization component is much greater than the intensity of the P polarization component, and then is split into two linearly polarized beams by the polarization beam splitter 6. The S light passes through the acousto-optic tunable filter 12 and the reflector 13 and then irradiates the spatial light modulator 14 to be modulated.

[0083] The controller is used to control the spatial light modulator 14 so that the phase modulation function of the spatial light modulator 14 is switched to:

[0084]

[0085] in, is the distance between a point on the beam and the optical axis, is the polar coordinate vector of the position in the section perpendicular to the optical axis of the beam and The angle of the axis.

[0086] Therefore, after phase modulation by the spatial light modulator 14, the electric vector intensity of the outgoing light beam can be expressed by the following formula:

[0087]

[0088] in, is the light beam incident on the spatial light modulator 14. The electric vector strength at is the outgoing light beam after phase modulation by the spatial light modulator 14. The electric vector intensity at , where i is an imaginary unit.

[0089] The principle of this application is as follows: by adjusting the saturated competitive light intensity, the effective PSF of two different SACs, namely SAC1 and SAC2, is obtained, and then the PSF of the circular ring is obtained by subtracting them, as shown below:

[0090]

[0091] Since the competitive light intensity values ​​of the two SACs are similar, the intensity distributions of the two PSFs are also similar. Figure 2 Middle (b) The effective PSF of SAC1 with lower competing light intensity has lower saturation effect; Figure 2 (a) The effective PSF of SAC2 with higher competing light intensity has a higher saturation effect; therefore, the full width at half maximum (FWHM) of SAC1 is slightly larger than the full width at half maximum of SAC2. By subtracting these two PSFs, we can get a donut-shaped PSF. d ,like Figure 2 Then, the PSF can be subtracted from the PSF of SAC1. d , and get a smaller PSF half-maximum full width value, such as Figure 2 As shown in (d), the equation of dSAC is derived as follows:

[0092]

[0093] Where r is the subtraction coefficient; the subtraction coefficient needs to be selected based on the PSF match, otherwise distortion may occur due to excessively negative values. Therefore, the r value should strike a balance between the achievable resolution and the negative values ​​caused by the difference. Methods for determining r include intensity weighted subtraction (IWS), which assigns a subtraction factor to each pixel based on the intensity distribution of the initial image, where r=0.8 is selected.

[0094] Figure 3 for Figure 2 The normalized results of the lateral position intensity of the four PSFs in the figure are as follows: “High light intensity SAC” corresponds to Figure 2 Middle (a), “Low light intensity SAC” corresponds to Figure 2 In (b), the “difference result” corresponds to Figure 2 In (c), “differential SAC” corresponds to Figure 2 (d). By difference calculation, Figure 3 The full width at half maximum (FWHM) of the middle curve "Differential SAC" is the smallest, achieving a further improvement in resolution.

[0095] Figure 4 (a) to (d) are the fluorescent nanoparticle imaging images of high-intensity SAC, low-intensity SAC, the difference between the two, and differential SAC, respectively. The scale bar is 500nm. From the comparison of the imaging images, we can see that Figure 4 The resolution of (d) is significantly improved, and the contrast of the image is also improved.

[0096] Those skilled in the art can understand that the above are only preferred examples of the invention and are not intended to limit the invention. Although the invention is described in detail with reference to the above examples, those skilled in the art can still modify the technical solutions recorded in the above examples or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, etc. made within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims

1. A differential nonlinear competitive super-resolution microscopy method, characterized in that: The following steps are involved: Step 1) adjusting the polarization of the collimated laser beam so that the intensity of the S polarization component is much greater than the intensity of the P polarization component; Step 2) dividing the laser beam processed in step 1) into two linearly polarized beams, namely, P light and S light; Step 3) performing phase modulation on the S light; Step 4) combining the phase-modulated S light and P light into one beam, and then converting it into left-handed circularly polarized light and right-handed circularly polarized light according to the polarization characteristics; Step 5) projecting the light beam converted into left-handed circularly polarized light and right-handed circularly polarized light onto the sample to scan the sample; Step 6) receiving the signal light emitted by the sample during scanning to obtain the first image; Step 7) By changing the intensity of the S light, the second image is obtained; Step 8) Perform differential processing on the results of step 6) and step 7), set the differential coefficient, return negative values ​​to zero, and finally obtain a super-resolution image.

2. The differential nonlinear competitive super-resolution microscopy method according to claim 1, characterized in that: In step 3), a spatial light modulator is used to phase modulate the S light.

3. The differential nonlinear competitive super-resolution microscopy method according to claim 1, characterized in that: During scanning in step 5), the phase-modulated P light is converged into a hollow light spot on the sample, and the S light is converged into a solid light spot on the sample.

4. A differential nonlinear competitive super-resolution microscopy device, comprising a light source and a sample stage for carrying a sample to be measured, characterized in that: The light source and the sample stage are provided in sequence: A first 1 / 2 wave plate, used to change the polarization characteristics of the light beam emitted by the light source; A polarization beam splitter, used to split the light beam after the polarization property is changed into two linearly polarized lights, wherein the two linearly polarized lights are P light and S light respectively; A spatial light modulator, used for phase modulating the S light; an acousto-optic tunable filter, used for controlling the intensity of the S light; The second 1 / 2 wave plate and the first 1 / 4 wave plate are used to change the polarization characteristics of the polarized light; A polarization beam combiner, used to combine the P light passing through the spatial light modulator and the S light passing through the acousto-optic modulator into one beam; The second quarter wave plate is used to change the polarization characteristics of the light in the light beam emitted by the polarization beam combiner; The sample scanning system is used to deflect the optical path of the light beams converted into left-handed circularly polarized light and right-handed circularly polarized light by the second quarter wave plate in sequence so as to project them onto the sample, and converge the phase-modulated P light into a hollow light spot on the sample, and converge the S light into a solid light spot on the sample to scan the sample; The detection system is used to collect the signal light emitted by the sample.

5. The differential nonlinear competitive super-resolution microscopy device according to claim 4, characterized in that: A single-mode optical fiber and a collimating lens for filtering the laser beam are arranged in sequence between the light source and the first 1 / 2 wave plate.

6. The differential nonlinear competitive super-resolution microscopy device according to claim 4 or 5, characterized in that: The spatial light modulator has a variable modulation function.

7. The differential nonlinear competitive super-resolution microscopy device according to claim 4, characterized in that: The sample scanning system includes a scanning galvanometer, a second 1 / 4 wave plate, a field lens and a microscope objective lens; The scanning galvanometer deflects the optical path of the light beam passing through the second 1 / 4 wave plate; The scanning lens and the field lens are used to focus and collimate the light beam emitted by the scanning galvanometer, respectively.

8. The differential nonlinear competitive super-resolution microscopy device according to claim 4, characterized in that: The detection system includes a beam splitter, a bandpass filter, a collection lens, a spatial filter and a detector; The beam splitter is arranged between the spatial light modulator and the sample scanning system; The bandpass filter is used to filter out stray light in the signal light emitted by the beam splitter; The collecting lens is used to focus the filtered signal light beam onto the detector; The spatial filter is used to perform spatial filtering on the signal light beam and is located at the focal plane of the collecting lens; The detector is used to detect the light intensity signal of the signal light beam.

9. The differential nonlinear competitive super-resolution microscopy device according to claim 8, characterized in that: When the sample to be measured is a fluorescent sample, the beam splitter uses a dichroic mirror.

10. The differential nonlinear competitive super-resolution microscopy device according to claim 8, characterized in that: The spatial filter adopts a pinhole or a multimode optical fiber. If a pinhole is adopted, the diameter of the pinhole is smaller than the diameter of an Airy disk.

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