Single molecule positioning method based on high-speed phase-locked detection

Abstract

The invention discloses a single molecule positioning method based on high-speed phase-locked detection, which can perform high-precision single molecule positioning and imaging on a fluorescent sample under the condition of using a single laser light source. Molecular excitation of a sub-diffraction limit region is generated by using nonlinear characteristics of excitation and fluorescence emission when a fluorescence sample is excited by laser, and meanwhile, positioning information is obtained by surrounding a single molecule of a region of interest by using an illumination light beam; and the laser intensity is continuously improved, and the positioning precision is also improved until sub-five-nanometer precision information is obtained. And the detection end adopts a high-speed lock-in amplifier to lock fluorescence signal information of characteristic frequency in real time, and meanwhile, the final signal-to-background ratio is also improved. Compared with traditional high-precision positioning based on double laser light sources, the method has the advantages of a single laser light source, does not need special fluorescent dye, does not need special matching between double-wavelength lasers, and has universality; and the damage caused by phototoxicity and photobleaching due to over-high illumination intensity is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of super-resolution and relates to a single-molecule positioning method based on high-speed phase-locked detection. Background Art

[0002] In order to resolve the position and structure information of fluorescent molecules within the diffraction limit, all lens-based fluorescence microscopy techniques must complete two steps: (1) activating the fluorescent dye to make adjacent fluorescent molecules distinguishable; (2) obtaining their high-precision coordinate information. As an imaging method beyond the diffraction limit, stimulated emission depletion super-resolution microscopy (STED) has attracted widespread attention since its proposal. The wonderful thing about STED is that the above two steps can be completed at one time, that is, through a "donut" shaped STED beam, the spontaneous emission of fluorescence is confined to a sub-diffraction size area.

[0003] The STED beam not only selects the fluorescent molecules that happen to be in the area, but also determines their location. The ability of the fluorescent spontaneous emission and the area determined by the STED beam can be well described by the "effective point spread function" (E-PSF), which is a Gaussian distribution with a full width at half maximum (FWHM): d = λ / (2NA ;Here, λ, NA, I and I s are the wavelength of the STED beam, the numerical aperture of the lens, the peak intensity, and the saturation intensity, respectively. Therefore, two fluorescent molecules with a distance greater than d can be separated by scanning the sample with coaxial excitation light and STED beam.

[0004] MINSTED microscopy, a technology that applies the idea of ​​STED to single-molecule localization, came into being, and is able to achieve spatial resolution of molecular size (1-3 nanometers). Compared with STED, MINSTED involves less bleaching and uses fewer photons than mainstream camera-based single-molecule localization techniques to achieve molecular-scale localization. However, this technology still requires dual-light source laser illumination for imaging, which leads to the complexity of its hardware; and the combination of excitation light and loss light relies on special dyes, which is not conducive to universal application. Summary of the invention

[0005] The present invention proposes a single-molecule localization method based on high-speed phase-locked detection, namely, minimum saturation competition localization imaging (MINSAC).

[0006] The present invention splits a single light source laser beam to obtain spatially modulated light and time modulated light. The two beams of light produce a saturated absorption competition effect on the sample surface, and use this effect to perform sub-five-nanometer single-molecule positioning of the pre-positioned fluorescent molecules. For each pixel point, a two-dimensional linked electro-optical deflector is used to draw a circle to obtain the change of its photon number over time, and its initial positioning is calculated; then the light intensity is continuously increased and the circle radius is reduced until the best positioning information is iterated. The number of photons required for the positioning information of each pixel point is demodulated by a high-speed lock-in amplifier.

[0007] Compared with the prior art, the present invention has the following beneficial technical effects:

[0008] (1) Only a single light source is needed to achieve sub-five-nanometer precision single-molecule localization;

[0009] (2) No special dyes are required and it has universal application;

[0010] (3) Low photobleaching and low phototoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A diagram of a specific implementation device for high-speed phase-locked detection of single-molecule localization;

[0012] Figure 2 Schematic diagram of the control principle for high-speed phase-locked detection of single-molecule positioning;

[0013] Figure 3 This is a schematic diagram of the principle of saturation absorption competition in this embodiment;

[0014] Figure 4 This is a high-precision positioning principle diagram of this embodiment;

[0015] Figure 5 This is the flowchart of the positioning algorithm strategy. DETAILED DESCRIPTION

[0016] 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.

[0017] A single molecule localization method based on high-speed phase-locked detection in an embodiment of the present application includes the following steps:

[0018] S1: Split the laser light emitted by the laser, and the two beams obtained are respectively subjected to time modulation and spatial phase modulation of the set frequency, corresponding to the solid excitation light I exc and hollow excitation light I hexc ;

[0019] The two photosynthetic beams compete for saturation absorption on the sample surface, resulting in limited fluorescence excitation.

[0020] S2: Pre-scan the observation area, obtain the best observation area and pixelate the area, and use each pixel as a single point for subsequent positioning.

[0021] S3: Positioning the pixelated area in step S2 point by point, specifically:

[0022] S301: Through single-point positioning, the combined beam on the sample surface is deflected in a two-dimensional rapid circular motion around the fluorescent molecule, with a radius of R i-1 (i is the number of times the position needs to be updated), generating fluorescence excitation;

[0023] The detector receives the emitted signal light, and demodulates the signal in real time at a set frequency to obtain the number of photons;

[0024] The initial position of the fluorescent molecule is obtained using the number of photons obtained.

[0025] S302: Improvement I hexc The light intensity is reduced to a radius of R. i , repeat the step S301 to update the position of the fluorescent molecule;

[0026] S303: repeat the steps in S302 until the number of acquired photons is lower than the threshold, stop the single point positioning process, and output the single point fluorescent molecule position information;

[0027] S304: Move to the next pixel (next point) and repeat steps S301 to S303 to obtain the fluorescent molecule position information of each pixel in the area.

[0028] S4: reconstructing the position information of the point-by-point positioning information obtained in step S3, and finally obtaining a sub-five-nanometer super-resolution image.

[0029] Furthermore, in step S1, the time modulation method is sinusoidal periodic or square wave periodic modulation, and the space modulation method is phase modulation, so as to generate vortex light.

[0030] Further, in step S1, the saturation absorption competition is solid excitation light I exc and hollow excitation light I hexc The interaction between the energy level excitations on the fluorescent sample surface, the former is a low-intensity beam, and the latter is a high-intensity beam.

[0031] Furthermore, in step S301, the two-dimensional rapid circular deflection adopts linkage cooperation between two-axis electro-optical deflectors, and its offset scale is within the diffraction limit.

[0032] Furthermore, in step S301, the signal light is detected by a photomultiplier tube, and the signal is demodulated by a high-speed lock-in amplifier.

[0033] Furthermore, in step S301, the method uses the minimum value of the center of the light beam as a movable reference coordinate to locate a single fluorescent molecule.

[0034] Furthermore, in step S302, the step of increasing the light intensity needs to be completed by an intensity modulator.

[0035] Furthermore, in the point-by-point positioning strategy, single-photon judgment must be performed first; if it is not a single photon, the illumination intensity must be increased to further bleach the area.

[0036] Furthermore, during the entire point-by-point positioning process, the drift correction module runs synchronously to ensure that the entire process is in a zero-drift state.

[0037] The principle of this application is as follows: Similar to the principle of STED breaking through the diffraction limit, the resolution of saturation absorption competition microscopy (SAC) is: d=λ / (2NA , where I hexc is the hollow excitation light intensity, I exc is the solid excitation light intensity, I s is the saturation light intensity; obviously, as I hexc The SAC imaging system can improve the resolution by increasing the intensity of the hollow light. Theoretically, as long as the hollow light intensity is high enough, extremely high resolution can be achieved.

[0038] However, practical resolving power is limited by several factors: imperfect central dark spots, photobleaching, beam center mismatch, and pulse mismatch (for pulse saturation competition mode). hexc About I s When the power density is 10 times higher, a high resolution of about 80 nanometers can be achieved. However, high power density will lead to aggravated side lobes. In actual implementation, it is believed that I hexc in I s One to five times is a moderate range in fluorescence imaging.

[0039] This application proposes a super-resolution fluorescence microscopy technology based on the saturation absorption competition (SAC) principle, called MINSAC, which can achieve molecular-level spatial resolution; its core is to use the central minimum value of the light beam as a movable reference coordinate to accurately locate a single fluorescent molecule.

[0040] The fluorescent molecules in the present invention are first transferred from an inactive state (off) to an active state (on), in which only one fluorescent molecule is activated at any given time, which means that its initial position is unknown on a diffraction-limited scale. The position of this fluorescent molecule can be determined by the central minimum of the SAC beam, because the central minimum of the SAC beam defines a coordinate to which the unknown fluorescent molecule coordinates can be associated.

[0041] MINSAC technology improves localization accuracy by adjusting the intensity of the hollow beam to "narrow" the effective point spread function (E-PSF). As the intensity of the hollow beam increases, the E-PSF becomes narrower, allowing for more precise localization. This approach not only reduces the number of photons required, but also reduces photobleaching and photoblinking. A key advantage of MINSAC technology is its ability to suppress background signals, which makes the imaging results almost free of background interference, thereby improving the clarity and contrast of the imaging.

[0042] The present application also provides a super-resolution microscopy device constructed based on the above method, such as Figure 1 As shown, the device includes: a laser light source 1, a polarization beam splitter 2, a light intensity modulator 3, a vortex phase plate 4, a first reflector 5, an electro-optic modulator 6, a polarization beam combiner 7, an x-direction electro-optical deflector 8, a y-direction electro-optical deflector 9, a dichroic mirror 10, a second reflector 11, an objective lens 12, a sample 13, a nano-piezoelectric displacement stage 14, a real-time drift correction module 15, a third reflector 16, a photomultiplier tube 17, and a phase-locked amplifier 18.

[0043] Specifically, the light beam emitted by the laser light source 1 is split into parallel polarized light and vertical polarized light by the polarization beam splitter 2, and enters the polarization beam combiner 7 through two first reflectors 5 respectively, and is combined into a beam of light. Then, it is two-dimensionally scanned by the x-direction electro-optical deflector 8 and the y-direction electro-optical deflector 9, and acts on the sample 13 through the dichroic mirror 10, the second reflector 11, and the objective lens 12. The generated fluorescence signal returns to the original path, passes through the objective lens 12, the second reflector 11, and the dichroic mirror 10, and is received by the photomultiplier tube 17, and finally the signal is demodulated by the lock-in amplifier 18.

[0044] The parallel polarized light mentioned above is controlled by the light intensity modulator 3 to control the light intensity, and is phase modulated by the vortex phase plate 4 to become a hollow light.

[0045] The vertically polarized light mentioned above is adjusted into a periodic timing light of a certain frequency by the electro-optic modulator 6, and the phase-locked amplifier 18 demodulates the signal at the same frequency.

[0046] The implementation device can be completed by several control modules, such as Figure 2 As shown in the figure, FPGA is a field programmable gate array and PC is the host. The beam deflection controller controls the two-dimensional deflection of the x-direction electro-optical deflector and the y-direction electro-optical deflector. The light source controller is responsible for the opening of the laser and the control of the light intensity. The signal detection controller is responsible for signal collection and signal demodulation. The anti-drift controller is responsible for high-precision real-time drift correction. The imaging control is responsible for the control of the objective lens. FPGA processes the data flow between the various controllers at high speed, including starting and stopping tasks, obtaining positioning information, updating positions, adjusting the excitation light intensity, etc.

[0047] Figure 3 is the principle of saturation absorption competition on the sample surface, I hexc is the intensity of the hollow excitation light, I exc is the intensity of the solid excitation light, E-PSF is the effective fluorescence emission area, S0 is the ground state of the fluorescent molecule, and S1 is the excited state. Through the single beam saturation absorption competition of laser illumination, the edge area of ​​the spot is hexc Much larger than I exc , resulting in saturated absorption and almost no effective fluorescence excitation; in the central region, I hexc Much smaller than I exc , which is unsaturated absorption and can achieve effective fluorescence excitation. This area can be used to acquire the photon number of pre-positioned fluorescent molecules.

[0048] Through the saturation absorption competition effect, the full width at half maximum (FWHM) of the beam can be obtained as follows: d = λ / (2NA , where I hexc is the light intensity of the hollow excitation beam, I s is the saturation light intensity, λ is the fluorescence wavelength, and NA is the numerical aperture of the imaging system.

[0049] The positioning accuracy of MINSAC is: σ = , where R is the radius of the circular path of the deflected light beam around the fluorescent molecules, and N is the number of photons obtained during positioning.

[0050] Obviously, by increasing I hexc , a smaller d value can be obtained; when the number of photons N is constant, σ can reach 1nm or even smaller, which is the theoretical basis for MINSAC to achieve sub-nanometer precision positioning.

[0051] Figure 4 The schematic diagram of high-precision positioning. In the figure, R represents the radius of the circular path of the fluorescent molecule by the deflection beam; i represents the number of iterative position updates required by bypassing the fluorescent molecule, and R i-1 is the radius of the circle drawn by the light beam at the initial position; by circling the fluorescent molecule, the updated position of the fluorescent molecule is obtained once each iteration, and the circling radius R i-1 Update to R i (R i+1 , R i+2 …); finally the best fluorescent molecule position is iterated.

[0052] Figure 5This is the flowchart of the positioning algorithm strategy. After starting the program, the observed area is pixelated, with 50-100nm as a pixel. Starting from the first pixel, the detector records the number of photons in the entire circumference and fits the molecular position; the center of the circle updates the position and improves I hexc The light intensity and the circle radius are reduced proportionally, and the molecular position is updated with the same strategy as above until it finally approaches the true fluorescence position, completing a set of iterative processes to obtain the single-point positioning of the pixel. Traverse the area, complete the above steps for each pixel, obtain the single-molecule positioning information of the entire area, output the single-point positioning information of all pixels, and output the super-resolution image, and the program ends.

[0053] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A single-molecule localization method based on high-speed phase-locked detection, characterized in that: The following steps are involved: Step S1: splitting the laser light emitted by the laser, and performing time modulation and spatial phase modulation on the obtained two beams to obtain corresponding solid excitation light and hollow excitation light; After the hollow excitation light and the solid excitation light combine, they compete for saturation absorption on the sample surface, resulting in limited fluorescence excitation. Step S2: pre-scanning the observation area to obtain the best observation area, and pixelating the area; Step S3: Positioning the pixelated area point by point; Step S4: reconstruct the position information of the obtained point-by-point positioning information, and finally obtain a sub-five-nanometer super-resolution image.

2. A single-molecule localization method based on high-speed phase-locked detection according to claim 1, characterized in that: The point-by-point positioning in step S3 is specifically as follows: Step S301: by single-point positioning, the combined beam on the sample surface is subjected to two-dimensional rapid circular deflection around the fluorescent molecules to generate fluorescence excitation; The detector receives the emitted signal light, and demodulates the signal light in real time to obtain the number of photons; Using the number of photons obtained, the initial position of the fluorescent molecule is obtained; Step S302: increasing the light intensity of the hollow excitation light, reducing the circumferential deflection radius, repeating the step S301, and updating the position of the fluorescent molecule; Step S303: repeat the steps in S302 until the number of acquired photons is lower than the threshold, stop the single point positioning process, and output the single point fluorescent molecule position information; Step S304: Move to the next pixel and repeat steps S301 to S303 to obtain the fluorescent molecule position information of each pixel in the area.

3. A single molecule localization method based on high-speed phase-locked detection according to claim 1 or 2, characterized in that: In step S1, the time modulation method is sinusoidal periodic or square wave periodic modulation, and the space modulation method is phase modulation.

4. A single-molecule localization method based on high-speed phase-locked detection according to claim 3, characterized in that: In step S1, a vortex phase plate is used to generate vortex light after spatial modulation.

5. A single-molecule localization method based on high-speed phase-locked detection according to claim 1 or 2, characterized in that: In step S1, the saturation absorption competition is the interaction between the energy level excitation of the solid excitation light and the hollow excitation light on the surface of the fluorescent sample, the former being a low-intensity light beam and the latter being a high-intensity light beam.

6. A single molecule localization method based on high-speed phase-locked detection according to claim 2, characterized in that: In step S301, the two-dimensional rapid circular deflection adopts the linkage cooperation between the two-axis electro-optical deflectors, and the offset scale is within the diffraction limit.

7. A single molecule localization method based on high-speed phase-locked detection according to claim 2 or 6, characterized in that: In step S301, the signal light is detected by a photomultiplier tube, and the real-time demodulation of the signal is performed by a high-speed lock-in amplifier.

8. A single molecule localization method based on high-speed phase-locked detection according to claim 2 or 6, characterized in that: In step S301, a single fluorescent molecule is positioned using the beam center minimum as a movable reference coordinate.

9. The single-molecule localization method based on high-speed phase-locked detection according to claim 2, characterized in that: In step S302, the light intensity of the hollow excitation light is increased by an intensity modulator.

10. A single-molecule localization method based on high-speed phase-locked detection according to claim 1 or 2, characterized in that: During the entire point-by-point positioning process, the drift correction module runs synchronously to ensure that the entire process is in a zero drift state.

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