An ultrahigh-resolution dark-field microscopy method based on nonlinear focal spot competition

Abstract

The application discloses a kind of super diffraction dark spot microscopic imaging methods based on nonlinear focal spot competition.The application carries out incoherent overlap by different wavelength of hollow light beam in focal point area, and nonlinear absorption competition of excitation photon occurs in the peripheral overlap area of fluorophore of double hollow light beam, finally generates super diffraction dark spot in focal plane, forms stable fluorescent signal in small size signal detection area, and extracts target signal by weak signal lock-in amplification technology.The application breaks through optical diffraction limit, so that it is possible to position the object to be observed in the diffraction limit region with high precision.The system requires low optical power and has high detection sensitivity, and the device structure is simple, suitable for high-resolution observation in the fields of biomedicine, nanomaterials and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of optical super-resolution imaging, and in particular to a method for realizing super-diffraction dark spot microscopic imaging through the nonlinear absorption competition effect of a dual-wavelength hollow light beam. Background Art

[0002] Under high-intensity illumination, fluorophores have a limit to their photon absorption. As the light intensity increases, the rate at which they absorb photons gradually increases until it reaches a peak and enters a saturated state. When the photon absorption rate of a fluorophore reaches saturation, the number of excited molecules will not increase linearly even if the light intensity is further increased. This nonlinear change in the absorption process is called nonlinear absorption. This property of nonlinear absorption has important applications in fields such as fluorophore localization, nanoparticle imaging, and super-resolution detection.

[0003] Traditional optical imaging is constrained by the Abbe diffraction limit, making it difficult to resolve structures smaller than half a wavelength (λ / 2). While existing super-resolution imaging techniques, such as stimulated emission depletion microscopy (SEDEM), can overcome these limitations, they rely on complex components such as polarization beam splitters and half-wave plates for polarization control. These techniques are also highly sensitive to temporal synchronization and spatial displacement, resulting in high system costs and difficulty in debugging. Furthermore, mechanisms based on stimulated emission are susceptible to interference from environmental noise, limiting their application in dynamic detection.

[0004] Existing technologies propose to use the incoherent superposition of two light beams to generate super-diffraction dark spots, but most of them are based on the principle of stimulated emission of radiation and fail to fully utilize the advantages of the nonlinear absorption competition effect. Summary of the Invention

[0005] The present invention provides a simple, high-resolution super-diffraction dark spot microscopy solution. Through the spatiotemporal incoherent superposition of dual-wavelength hollow light beams and based on the nonlinear absorption competition effect of fluorophore excitation photons, the effective fluorescence signal area is compressed to a super-diffraction limit scale. There is no need to rely on complex components such as polarization state control and spatiotemporal precision calibration in traditional technologies, which significantly improves the practicality and system stability of super-resolution imaging.

[0006] A super-diffraction dark spot microscopy imaging method based on nonlinear focal spot competition of the present invention comprises the following steps:

[0007] S1. Dual-wavelength laser output, using two pulsed lasers to output beams separately;

[0008] S2 dual beam modulation, respectively, the two beams are collimated, spatially modulated, polarization modulated and frequency modulated to generate a donut-shaped double hollow beam;

[0009] S3. Spatiotemporal overlap, through the temporal synchronization regulation mechanism and two-dimensional scanning, realize the synchronization of the double hollow light beams in the time domain and the overlap in the spatial domain;

[0010] S4. High focusing scanning, through the focusing system, focus the double hollow light beams to the sample plane and realize the fast two-dimensional focusing scanning;

[0011] S5. Signal detection, through the optical light splitting component, carry out the spectral separation and light path guidance of the fluorescence signal, utilize the optical converging component to focus the separated signal, then transmit the signal to the photoelectric detector through the optical transmission medium, finally carry out the amplification processing and collection of the detected signal by the signal demodulation equipment, realize the efficient detection of the fluorescence signal.

[0012] Compared with the prior art, the present application has the following significant technical advantages:

[0013] Super-resolution performance: the peripheral size of the focal spot breaks through the diffraction limit and can reach below 50nm, and the resolution is improved to 1 / 4 of the traditional method;

[0014] System simplification: omitting components such as polarization beam splitter and half-wave plate, the optical path structure is relatively simplified, and the required laser power is smaller, and the cost is greatly reduced;

[0015] High signal-to-noise ratio: using weak signal lock amplification technology to suppress background noise, improve signal-to-noise ratio, and improve weak signal detection capability. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The flow chart of the super-diffraction dark spot microscopic imaging method of the embodiment;

[0017] Figure 2 The optical path schematic diagram of the system in the embodiment;

[0018] Figure 3 The principle diagram of the super-diffraction dark spot microscopic imaging based on nonlinear focal spot competition in the embodiment;

[0019] Figure 4 The simulation result diagram of the array distribution sample in the embodiment;

[0020] Figure 5 The sample diagram in the embodiment;

[0021] Figure 6 The effect diagram of the stimulated emission depletion microscopic imaging technology in the embodiment;

[0022] Figure 7 The effect diagram of the super-diffraction dark spot microscopic imaging method based on nonlinear focal spot competition in the embodiment. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the protection scope of the present application.

[0024] As shown in the embodiment of the present application, a super-diffraction dark spot microscopic imaging method based on nonlinear focal spot competition comprises the following steps: Figure 1

[0025] S1. Dual-wavelength hollow beam preparation: two pulsed lasers are used to output light beams, and the two light beams are collimated, spatially modulated, polarized and frequency modulated to generate a doughnut-shaped dual-hollow beam;

[0026] S2. Time and space synchronous superposition: time sequence synchronization control mechanism and two-dimensional scanning are used to realize time domain synchronization and spatial domain overlap of the dual-hollow beam;

[0027] S3. High focusing scanning: the dual-hollow beam is focused to the sample plane by a focusing system and rapid two-dimensional focusing scanning is realized, and nonlinear absorption competition occurs between the two light beams;

[0028] S4. Signal detection and processing: the optical splitting component is used to guide the optical path of the fluorescence signal, the optical focusing component is used to focus the separated signal, the signal is transmitted to the photodetector through the optical transmission medium, and finally the signal demodulation device is used to amplify, process and collect the detected signal, thereby realizing efficient detection of the fluorescence signal;

[0029] S5. System control and parameter adjustment: signal synchronization and parameter monitoring are used to realize precise control of the system.

[0030] Further, in step S1, the dual-wavelength hollow beam preparation comprises the following steps:

[0031] S101. After the laser pulse light beam is collimated into a parallel light beam by a collimating lens, it passes through a vortex phase plate to generate a doughnut-shaped hollow beam, and the peripheral light intensity conforms to a Laguerre-Gaussian distribution;

[0032] S102. The dual-wavelength hollow beam is respectively polarized and frequency modulated;

[0033] S103. The function generator outputs an electrical signal with a reference frequency f0 to drive an electro-optical modulator, which is time modulated through electro-optical effect to generate a light beam with stable pulse width and adjustable frequency;

[0034] The excitation light beam and the nonlinear absorption competition light beam are modulated by two specific frequencies f1 and f2, and the frequency f1 is used as the reference frequency of the demodulated target fluorescence signal. ​

[0035] Further, in step S2, the double hollow beams are adjusted by the mirror to adjust the optical path, the coaxial transmission is realized by the dichroic mirror, the time delay of the 561 nm beam is accurately adjusted by the time delay scheme of the synchronous pulse, the photodetector signals of the two beams are monitored in real time by the data acquisition card, and the time overlap error is ensured to be less than 10 ps, that is, within 1 / 10 of the pulse full width at half maximum;

[0036] Further, in step S2, the spatial calibration of the light beam is performed by the galvanometer-based scanning mirror, the galvanometer angle is controlled by the computer program, the central axis deviation of the hollow excitation light beam and the hollow nonlinear absorption competition light beam is controlled in a small range, and the concentric overlapping doughnut-shaped light spots are formed on the focal plane, and the light intensity uniformity in the overlapping area is greater than 95%.

[0037] Further, in step S3, the coaxial double hollow beams are focused to the sample plane by the objective lens to form a hollow focusing light spot, in the central low-intensity area, the fluorophore does not completely transition to the excited state, and in the peripheral area, the two light beams undergo nonlinear absorption competition; when the intensity of the hollow nonlinear absorption competition light beam exceeds the threshold value, fluorescence saturation occurs in the peripheral area, and the signal is suppressed, and fluorescence saturation does not occur in the central low-intensity area, forming an ultradiffraction dark spot with a peripheral size of less than 50 nm;

[0038] Further, in step S3, the galvanometer, the scanning mirror head and the tube lens form a two-dimensional scanning system, the raster scanning is performed at a frequency of 100 Hz, the scanning range is 10 μm×10 μm, the step is 20 nm, the rapid scanning of the sample is realized, and the imaging resolution is ensured.

[0039] Further, in step S4, the signal detection and processing technology comprises the following steps:

[0040] S401. After the fluorescence signal is collected by the objective lens, the fluorescence signal is reflected by the dichroic mirror, the stray light is filtered by the optical filter, and then focused to the pinhole on the multimode optical fiber by the collection lens, the non-focal plane signal is filtered, the photomultiplier converts the light signal into an electrical signal, and the lock-in amplifier performs homodyne detection by using the same frequency reference signal (frequency f0) provided by the function generator, and the fluorescence signal of the hollow excitation light beam is selectively demodulated and processed;

[0041] S402. By means of accurate frequency matching and signal processing mechanism, the fluorescence signal related to the hollow nonlinear absorption competition light beam is identified as a non-target signal, and the non-target signal is effectively suppressed or removed by using the filtering technology, so that the target signal is extracted and separated with high signal-to-noise ratio.

[0042] Further, in step S5, the system control and parameter adjustment technology comprises the following steps:

[0043] S501. The function generator provides a synchronous reference signal for the electro-optical modulator and the lock-in amplifier, and the data acquisition card monitors system parameters (such as light intensity, time delay error) in real time and transmits them to the computer;

[0044] S502. The computer serves as the control core, controls the scanning path of the galvanometer, the parameters of the electro-optical modulator and the time delay calibration through the program, and simultaneously demodulates the fluorescence signal output by the lock-in amplifier and reconstructs the image.

[0045] To sum up, the application solves the problems of complex system and low imaging precision in the prior art by inducing nonlinear absorption competition through the intensity difference of the double hollow light beams.

[0046] Further, to realize the above method, the embodiment also provides a light path schematic diagram of the system. Figure 2 As shown in the figure, in the embodiment, a super-diffraction dark spot microscopic imaging system based on nonlinear focal spot competition includes a 532nm laser diode 1, a first single-mode optical fiber 2, a collimating lens 3, a first reflecting mirror 4, a first vortex phase plate 5, a first Glan prism 6, a first electro-optical modulator 7, a second reflecting mirror 8, a first dichroic mirror 9, a 561nm laser diode 10, a second single-mode optical fiber 11, a collimating lens 12, a third reflecting mirror 13, a synchronous pulse time delay scheme 14, a second vortex phase plate 15, a second Glan prism 16, a second electro-optical modulator 17, a second dichroic mirror 18, a fourth reflecting mirror 19, a fifth reflecting mirror 20, a scanning mirror based on a galvanometer 21, a sixth reflecting mirror 22, a scanning lens 23, a tube lens 24, an achromatic quarter-wave plate 25, an objective lens 26, a sample plane 27, a seventh reflecting mirror 28, an eighth reflecting mirror 29, a ninth reflecting mirror 30, a filter 31, a collection lens 32, a multimode optical fiber 33, a photomultiplier tube 34, a lock-in amplifier 35, a function generator 36, a voltage amplifier 37, a data acquisition card 38, and a computer 39.

[0047] The first single-mode optical fiber 2, the collimating lens 3, the first reflecting mirror 4, the first vortex phase plate 5, the first Glan prism 6, the first electro-optical modulator 7 and the second reflecting mirror 8 are sequentially located on the optical axis of the light beam emitted by the 532nm laser diode 1. The first single-mode optical fiber 11, the collimating lens 12, the third reflecting mirror 13, the synchronous pulse time delay scheme 14, the second vortex phase plate 15, the second Glan prism 16 and the second electro-optical modulator 17 are sequentially located on the optical axis of the light beam emitted by the 561nm laser diode 10; the light beam reflected by the second reflecting mirror 8 and the light beam emitted by the second electro-optical modulator 17 converge on the first dichroic mirror 9.

[0048] Among them, the fourth reflector 19, the fifth reflector 20, the galvanometer-based scanning mirror 21, the sixth reflector 22, the scanning lens 23, the tube lens 24, the achromatic quarter-wave plate 25, the objective lens 26 and the sample plane 27 are sequentially located above the optical axis of the light beam transmitted by the second dichroic mirror 18.

[0049] Among them, the seventh reflector 28, the eighth reflector 29, the ninth reflector 30, the filter 31, the collecting lens 32, the multimode optical fiber 33 and the photomultiplier tube 34 are sequentially located on the optical axis of the light beam reflected by the second dichroic mirror 18.

[0050] The photomultiplier tube 34 is connected to the lock-in amplifier 35 ; the voltage amplifier 37 is connected to the first electro-optic modulator 7 and the second electro-optic modulator 17 at the same time.

[0051] In the above system, the multimode optical fiber 33 can be further used as a filtering pinhole.

[0052] Further, based on Figure 2 The specific method for performing super-diffraction dark spot microscopy imaging with the system shown is as follows:

[0053] The light beam emitted by the 532nm laser diode 1 is transmitted through the first single-mode optical fiber 2, collimated by the collimating lens 3, deflected by the first reflector 4, and generated into a hollow excitation beam 1 by the first vortex phase plate 5. The light beam is then polarization modulated by the first Glan prism 6 and frequency modulated by the first electro-optical modulator 7 (driven by the function generator 37 to load the frequency f1), and then reflected by the second reflector 8 to the first dichroic mirror 9.

[0054] At the same time, the beam emitted by the 561 nm laser diode 10 is transmitted through a second single-mode fiber 11, collimated by a collimating lens 12, deflected by a third mirror 13, time-delayed by a synchronous pulse time delay scheme 14, and generated by a second vortex phase plate 15 to form a hollow nonlinear competing beam 2. After polarization modulation by a second Glan prism 16 and frequency modulation by a second electro-optical modulator 17 (with a loading frequency f2), the beam is emitted to the first dichroic mirror 9 for coaxial superposition with the hollow excitation beam 1. The coaxial beam is transmitted through the second dichroic mirror 18, deflected by a fourth mirror 19 and a fifth mirror 20 in sequence to a galvanometer-based scanning mirror 21 (controlled by a computer 39), and then passed through a sixth mirror 22, a scanning lens 23, a tube lens 24, and an achromatic quarter-wave plate 25. It is focused by an objective lens 26 onto a sample plane 27 to form a spatially overlapping spot. At sample plane 27, nonlinear absorption competition occurs between the hollow excitation beam and the hollow nonlinear competing beam in the overlapping region. Excitation of the fluorophore's peripheral region is suppressed due to photon absorption saturation, while linear excitation forms a super-diffraction dark spot in the central region.

[0055] The fluorescence signal is collected by the objective lens 26, reversely passes through the achromatic quarter-wave plate 25, the tube lens 24, the scanning lens 23, the sixth mirror 22, the galvanometer-based scanning mirror 21, the fifth mirror 20, the fourth mirror 19, is reflected by the second dichroic mirror 18 to the seventh mirror 28, the eighth mirror 29, the ninth mirror 30, is filtered by the filter 31, is focused by the collection lens 32 to the multimode optical fiber 33 (a pinhole filter is used to remove the non-focal plane signal), is converted into an electrical signal by the photomultiplier tube 34, is processed by the lock-in amplifier 35 (a reference frequency f0 of the function generator 36 is used to lock and amplify the frequency f1 signal and filter the frequency f2 signal), in the process, the function generator 36 provides a synchronous reference signal for the lock-in amplifier 35, and the voltage amplifier 37 is used to amplify the electrical signal, so that the intensity of the electrical signal meets the working requirements of the first electro-optical modulator 7 and the second electro-optical modulator 17, the transmission efficiency is improved, the driving capacity is enhanced, and finally the signal is collected by the data acquisition card 38 to the computer 39 for image reconstruction.

[0056] Figure 3 It is a schematic diagram of the nonlinear focal spot competition-based super-diffraction dark spot microscopic imaging in the embodiment. Wherein (a), (b) and (c) are respectively a hollow excitation light beam, a hollow nonlinear competition light beam and a super-diffraction dark spot. The two hollow light beams in the shape of a donut are overlapped in the time domain and the spatial domain, and are incoherently superimposed; the hollow nonlinear absorption competition light beam competes with the hollow excitation light beam, the double hollow light beams compete in the excitation photon absorption in the overlapping region, the peripheral region of the fluorophore is excited to a saturated state by the high-intensity competition light, and the central region still maintains a linear relationship with the excitation light intensity, and finally a peripheral signal competition region and an effective signal region are formed, and at the same time the effective signal region is compressed to a super-diffraction scale, generating a super-diffraction dark spot.

[0057] Figure 4 It is a simulation result diagram of the array distribution sample in the embodiment. Wherein (a) is a 19x19 dot array sample with a pitch of 100 nm; (b) is an imaging simulation result of (a) by stimulated emission depletion microscopy; (c) is a simulation result of the nonlinear focal spot competition-based super-diffraction dark spot microscopic imaging method in the embodiment. The middle regions of (b) and (c) are analyzed for intensity distribution, corresponding to (d) and (e) respectively. Obviously, (e) has a higher intensity contrast, that is, a stronger detail resolution, relative to (d).

[0058] Figure 5 It is a sample diagram in the embodiment, Figure 6 It is an effect diagram of the conventional stimulated emission depletion super-resolution microscopic imaging technology in the embodiment, Figure 7 It is an effect diagram of the nonlinear focal spot competition-based super-diffraction dark spot microscopic imaging method in the embodiment. Compared with the stimulated emission depletion microscope imaging technology, the image quality and the resolution of the nonlinear focal spot competition-based super-diffraction dark spot microscopic imaging method are significantly improved. The intensity distribution of the middle region of (b) and (c) is analyzed, corresponding to (d) and (e) respectively.Figure 5 , Figure 6 and Figure 7 The wireframe identified areas of the image that were not in the wireframe of the image of the same area taken by the microscope imaging method used in the present embodiment.

Claims

1. A super-diffraction dark spot microscopy imaging method based on nonlinear focal spot competition, characterized in that: The following steps are involved: S1. Dual-wavelength laser output, using two pulsed lasers to output beams separately; S2 dual beam modulation, respectively, the two beams are collimated, spatially modulated, polarization modulated and frequency modulated to generate a donut-shaped double hollow beam; S3. Spatiotemporal overlap: Through a time-series synchronization control mechanism and two-dimensional scanning, dual hollow beams are synchronized in the time domain and overlapped in the spatial domain. S4. High-focus scanning: Through the focusing system, the dual hollow beams are focused onto the sample plane and achieve rapid two-dimensional focused scanning; S5. Signal detection: Optical spectrometers separate and guide the fluorescence signal. Optical convergence components focus the separated signal, which is then transmitted to a photodetector via an optical transmission medium. Finally, a signal demodulator amplifies, processes, and collects the detected signal, achieving efficient fluorescence signal detection.

2. The method according to claim 1, characterized in that The dual-beam modulation comprises the following steps: The phase modulation optical element modulates the collimated two light beams into a donut-shaped hollow beam. The central intensity of the generated beam is zero, and the peripheral intensity has a Gaussian envelope distribution. The first beam is modulated into a hollow excitation beam, and the second beam is modulated into a hollow nonlinear absorption competition beam. The peak intensities of the first and second beams are adjusted so that the peak intensity of the second beam is significantly higher than that of the first beam. The two laser beams are intensity modulated using sinusoidal wave signals f1 and f2 so that the light intensities of the two laser beams exhibit periodic time-varying characteristics.

3. The method according to claim 2, characterized in that The spatiotemporal overlap is achieved by using a timing synchronization control mechanism to achieve temporal overlap of the two light beam pulses, and by two-dimensional scanning to achieve incoherent superposition of the two light beams in the spatial domain. The high intensity of the second light beam causes the two light beams to compete for nonlinear absorption of excitation photons in the overlapping area outside the fluorophore. After the photon absorption reaches its peak, the fluorescence signal no longer changes with the light intensity.

4. The method according to claim 3, characterized in that The time overlap error is less than 10 ps, ​​that is, within 1 / 10 of the pulse half-width.

5. The method according to claim 3, characterized in that The incoherent superposition is specifically: based on the scanning mirror of the galvanometer, the light beam is spatially calibrated, and the angle of the galvanometer is controlled so that the central axis deviation of the first light beam and the second light beam is controlled within a set threshold, thereby forming concentrically superimposed donut-shaped light spots in the focal plane, and the light intensity uniformity in the overlapping area is greater than 95%.

6. The method according to any one of claims 3 to 5, characterized in that The two-dimensional scanning system consists of a galvanometer, a scanning lens, and a tube lens. It performs raster scanning at a frequency of 100 Hz, with a scanning range of 10 μm × 10 μm and a step length of 20 nm, enabling rapid scanning of samples and ensuring imaging resolution.

7. The method according to claim 1, characterized in that During the high-focus scanning process, the coaxial dual hollow light beams are focused by the objective lens onto the sample plane to form a hollow focused light spot. In the central low-intensity area, the fluorophore has not completely transitioned to the excited state, while in the peripheral area, the two beams undergo nonlinear absorption competition.

8. The method according to claim 7, characterized in that When the intensity of the second light beam exceeds the threshold, fluorescence saturation occurs in the peripheral area and the signal is suppressed. No fluorescence saturation occurs in the central low-intensity area, forming a super-diffraction dark spot with an outer size of less than 50nm.

9. The method according to claim 2, characterized in that During the signal detection process, the photodetector is connected to a signal demodulation device. The signal demodulation device is based on the reference frequency provided by the signal source device. During the fluorescence imaging process, the signal demodulation device filters out the signal frequency f2 generated by the second light beam through filtering and frequency discrimination mechanisms, and accurately extracts the fluorescence signal frequency f1 excited by the first light beam.

10. The method according to claim 9, characterized in that It also includes the process of collecting the fluorescence signal through the objective lens, reflecting it through a dichroic mirror, filtering out stray light through a filter, and then focusing it to a pinhole on the multimode optical fiber by a collecting lens to filter out out-of-focus plane signals; The photomultiplier tube converts the optical signal into an electrical signal, and the lock-in amplifier uses the same-frequency reference signal provided by the function generator to perform homodyne detection and selectively demodulate the fluorescence signal of the first light beam.

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