Super-diffraction dark spot microscopic imaging method based on nonlinear focal spot competition

Abstract

The invention discloses a super-diffraction dark spot microscopic imaging method based on nonlinear focal spot competition. The hollow light beams with different wavelengths are subjected to incoherent overlapping in a focus area, the double hollow light beams generate excitation photon nonlinear absorption competition in a fluorophore peripheral overlapping area, super-diffraction dark spots are finally generated in a focal plane, and stable fluorescence signals are formed in a small-size signal detection area. A target signal is extracted through a weak signal locking amplification technology. The optical diffraction limit is broken through, so that high-precision positioning of the to-be-observed object in the diffraction limit area becomes possible. The optical power needed by the system is low, the detection sensitivity is high, the device structure is simple, and the system is suitable for high-resolution observation in the fields of biomedicine, nano materials 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 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.

[0010] 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;

[0011] 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.

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

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

[0014] System simplification: Components such as polarization beam splitters and half-wave plates are omitted, the optical path structure is relatively simplified, the required laser power is small, and the cost is greatly reduced;

[0015] High signal-to-noise ratio: Weak signal lock-in amplification technology is used to suppress background noise, improve the signal-to-noise ratio, and enhance weak signal detection capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Flow chart of the super-diffraction dark spot microscopy method of this embodiment;

[0017] Figure 2 Schematic diagram of the optical path of the system in this embodiment;

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

[0019] Figure 4 : is a simulation result diagram of the array distribution sample in this embodiment;

[0020] Figure 5 This is a sample diagram in this embodiment;

[0021] Figure 6 This is a diagram showing the effect of the stimulated emission depletion microscopy imaging technology used in this embodiment;

[0022] Figure 7 This is a diagram showing the effect of the super-diffraction dark spot microscopy imaging method based on nonlinear focal spot competition in this embodiment. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

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

[0025] S1. Dual-wavelength hollow beam preparation: Two pulsed lasers are used to output separate beams, which are then collimated, spatially modulated, polarized, and frequency modulated to generate donut-shaped dual hollow beams.

[0026] S2. Spatiotemporal synchronization superposition: 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.

[0027] S3. High-focus scanning: Through the focusing system, the dual hollow beams are focused onto the sample plane and achieve rapid two-dimensional focused scanning, resulting in nonlinear absorption competition between the two beams.

[0028] S4. Signal detection and processing: The fluorescence signal is guided by an optical splitter, focused by an optical convergence component, and 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.

[0029] S5. System control and parameter adjustment: precise control of the system is achieved through signal synchronization and parameter monitoring.

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

[0031] S101. After being collimated by a collimating lens into a parallel beam, the laser pulse beam passes through a vortex phase plate, generating a donut-shaped hollow beam with an outer intensity that conforms to a Laguerre-Gaussian distribution.

[0032] S102. Polarization modulation and frequency modulation are performed on the dual-wavelength hollow beam respectively;

[0033] S103. The function generator outputs an electrical signal of a reference frequency f0 to drive the electro-optic modulator, which performs time modulation through the electro-optic effect to generate a beam with a stable pulse width and adjustable frequency;

[0034] The excitation beam and the nonlinear absorption competition beam are modulated respectively using two specific frequencies f1 and f2, and the frequency f1 is used as the reference frequency for demodulating the target fluorescence signal.

[0035] Furthermore, in step S2, the optical paths of the two hollow beams are adjusted by a reflector, and coaxial transmission is achieved through a dichroic mirror. A time delay scheme of a synchronized pulse is used to accurately adjust the time delay of the 561nm beam. The photodetector signals of the two beams are monitored in real time by a data acquisition card to ensure that the time overlap error is less than 10ps, that is, within 1 / 10 of the pulse half-maximum width.

[0036] Furthermore, in step S2, the light beam is spatially calibrated based on the scanning mirror of the galvanometer, and the galvanometer angle is controlled by a computer program so that the central axis deviation of the hollow excitation beam and the hollow nonlinear absorption competition beam is controlled within a small range, forming concentrically superimposed donut-shaped light spots in the focal plane, and the light intensity uniformity in the overlapping area is greater than 95%.

[0037] Furthermore, in step S3, 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 region, the fluorophore has not completely transitioned to the excited state, while in the peripheral region, the two light beams compete for nonlinear absorption. When the intensity of the hollow nonlinear absorption competition beam exceeds a threshold, fluorescence saturation occurs in the peripheral region, the signal is suppressed, and fluorescence saturation does not occur in the central low-intensity region, forming a super-diffraction dark spot with a peripheral size of less than 50 nm.

[0038] Furthermore, in step S3, the galvanometer, scanning lens and tube lens form a two-dimensional scanning system, which 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, to achieve rapid scanning of the sample and ensure imaging resolution.

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

[0040] S401. The fluorescence signal is collected by the objective lens, reflected by a dichroic mirror, and filtered through a filter to remove stray light. It is then focused by a collecting lens onto a pinhole in a multimode optical fiber, filtering out out-of-focus signals. A photomultiplier tube converts the optical signal into an electrical signal. A lock-in amplifier uses a co-frequency reference signal (frequency f0) provided by a function generator for homodyne detection, selectively demodulating the fluorescence signal from the hollow excitation beam.

[0041] S402. Through precise frequency matching and signal processing mechanisms, the fluorescence signal associated with the hollow nonlinear absorption competing beam is identified as a non-target signal, and filtering technology is used to effectively suppress or remove it, thereby achieving high signal-to-noise ratio extraction and separation of the target signal.

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

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

[0044] S502. The computer serves as the control core, controlling the galvanometer scanning path, electro-optic modulator parameters, and time delay calibration through a program, while also demodulating the fluorescence signal output by the lock-in amplifier and reconstructing the image.

[0045] In summary, the present invention induces nonlinear absorption competition through the intensity difference of the dual hollow light beams, thereby solving the problems of complex system and low imaging accuracy in the prior art.

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

[0047] Among them, the first single-mode optical fiber 2, the collimating lens 3, the first reflector 4, the first vortex phase plate 5, the first Glan prism 6, the first electro-optic modulator 7, and the second reflector 8 are sequentially located on the optical axis of the output light beam of the 532nm laser diode 1. The first single-mode optical fiber 11, the collimating lens 12, the third reflector 13, the time delay scheme 14 for the synchronization pulse, the second vortex phase plate 15, the second Glan prism 16, and the second electro-optic modulator 17 are sequentially located on the optical axis of the output light beam of the 561nm laser diode 10. The light beam reflected by the second reflector 8 and the light beam emitted by the second electro-optic 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 and then passes back through the achromatic quarter-wave plate 25, the tube lens 24, the scanning lens 23, the sixth reflector 22, the galvanometer-based scanning mirror 21, the fifth reflector 20, and the fourth reflector 19. It is then reflected by the second dichroic mirror 18 to the seventh reflector 28, the eighth reflector 29, and the ninth reflector 30. It is filtered by the filter 31 and focused by the collecting lens 32 onto the multimode optical fiber 33 (the pinhole filters out the out-of-focal plane signal). It is then converted into an electrical signal by the photomultiplier tube 34 and processed by the phase-locked amplifier 35 (which locks the amplified frequency f1 signal based on the reference frequency f0 of the function generator 36 and filters out the frequency f2 signal). During this process, the function generator 36 provides a synchronization reference signal for the phase-locked amplifier 35, and the voltage amplifier 37 is used to amplify the electrical signal to ensure that its intensity meets the working requirements of the first electro-optical modulator 7 and the second electro-optical modulator 17, thereby improving transmission efficiency and enhancing driving capability. Finally, the signal is collected by the data acquisition card 38 and transmitted to the computer 39 for image reconstruction.

[0056] Figure 3 The schematic diagram of the super-diffraction dark spot microscopy based on nonlinear focal spot competition in this embodiment is shown in Figure 1. (a), (b), and (c) are the hollow excitation beam, the hollow nonlinear competition beam, and the super-diffraction dark spot, respectively. Two donut-shaped hollow beams overlap in the time and space domains, and are incoherently superimposed. The hollow nonlinear absorption competition beam competes with the hollow excitation beam, and the two hollow beams compete for excitation photon absorption in the overlapping area. The peripheral area of ​​the fluorophore is excited to a saturation state by the high-intensity competition light, while the central area still maintains a linear relationship with the excitation light intensity. Finally, a peripheral signal competition area and an effective signal area are formed. At the same time, the effective signal area is compressed to a super-diffraction scale, generating a super-diffraction dark spot.

[0057] Figure 4 The following are simulation results for the array distribution sample in this embodiment. (a) is a 19×19 dot matrix sample with a spacing of 100nm; (b) is the simulated imaging result of (a) using stimulated emission depletion microscopy; and (c) is the simulation result of the super-diffraction dark spot microscopy imaging method based on nonlinear focal spot competition in this embodiment. The intensity distribution analysis of the middle region between (b) and (c) corresponds to (d) and (e), respectively. Clearly, (e) has higher intensity contrast than (d), that is, stronger detail resolution.

[0058] Figure 5 is a sample diagram in this embodiment, Figure 6 This is the effect diagram of the traditional stimulated emission depletion super-resolution microscopy technology used in this embodiment. Figure 7 This is a diagram showing the effect of the super-diffraction dark spot microscopy imaging method based on nonlinear focus competition in this embodiment. Compared with stimulated emission depletion microscopy imaging technology, the image quality and resolution of the super-diffraction dark spot microscopy imaging method based on nonlinear focus competition are significantly improved. Figure 5 、 Figure 6 and Figure 7 By comparing the area marked by the wireframe, it is found that the microscopic imaging method used in this embodiment can distinguish more details.

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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