A super-resolution imaging system for non-fluorescent molecules based on photothermal relaxation localization microscopy.

Abstract

The present invention discloses a non-fluorescent molecular super-resolution imaging system based on photothermal relaxation localization microscopy, which includes a pump light source, a detection light source, a microscope, a photodetector, a multi-harmonic signal extraction device, and an imaging processing device. The detection light, modulated by the photothermal effect of the target sample and the dissipation of photothermal energy, undergoes multi-harmonic demodulation, and then super-resolution imaging is performed based on the high-harmonic signals extracted from the frequency domain. This method enables super-resolution imaging of light-absorbing molecules or structures without fluorescent labels, fundamentally resolving several limitations of conventional super-resolution imaging, such as photobleaching, labeling efficiency, and labeling selectivity of fluorescent molecules. Furthermore, it fundamentally eliminates various limitations of super-resolution fluorescence, such as the need for specially designed fluorescent molecules and photodamage caused by high-power lasers.

Description

Technical Field

[0001] The present invention belongs to the field of super-resolution imaging, and particularly relates to a non-fluorescent molecular super-resolution imaging system based on a photo-thermal relaxation localization microscope.

Background Art

[0002] Optical imaging beyond the diffraction limit provides a structural and dynamic view for in-situ analysis and has found fruitful applications in many fields. The most widely adopted imaging technology, fluorescence-based super-resolution microscopy, breaks through the optical diffraction limit, improves resolution, and accurately locates the positions of fluorescent molecules through stimulated emission depletion (STED) mechanisms or stochastic optical reconstruction (PALM or STORM). However, such technologies require special fluorescent molecules and complex collection methods. To solve this problem, super-resolution imaging technologies based on the saturation effect of fluorescence, such as saturation excitation microscopy (SAX), have been developed, improving resolution by demodulating higher-order harmonics in intensity modulation. However, the most fundamental problem with these super-resolution imaging methods arises from the dependence on fluorescent labels, and their development is limited by the cytotoxicity, labeling efficiency, and specificity of fluorescent labels.

[0003] Therefore, label-free imaging techniques have become a major technological goal. To this end, considerable effort has been made towards super-resolution imaging of endogenous biomolecules or materials. Similar to the STED mechanism, the loss of electronically excited states has been used to improve the resolution of material imaging, such as photoacoustic imaging by material saturation transient absorption (STAN) and heterogeneous bleaching of absorbing molecules. Structured pump-probe microscopy has demonstrated another structured illumination method. Simultaneously, nonlinear responses caused by heterogeneous intensity at the focal point have been used to improve resolution, such as in nonlinear photoacoustic microscopy (NL-PAM) and nonlinear photothermal imaging (NI-PTM). However, most of these techniques are based on signal saturation effects and photothermal nonlinearity, thus limiting the types of molecules that can be imaged, requiring high-power pulsed lasers, and hindering the widespread application of the above techniques.

[0004] In addition to label-free imaging using electron absorption, vibrational spectroscopy, such as infrared (IR) spectroscopy and Raman spectroscopy, provides molecular structure information based on intrinsic molecular state vibrations and is used to transition to label-free super-resolution imaging with molecular selectivity, such as structured light illumination Raman microscopy. Over the past decade, super-resolution imaging based on coherent Raman scattering, including high-order coherent anti-Stokes Raman scattering (HO-CARS), saturated coherent anti-Stokes Raman scattering, and saturated stimulated Raman scattering (SSRS), has been demonstrated. However, one of the most fundamental limitations of Raman spectroscopy is the extremely weak Raman effect. Coherent Raman scattering super-resolution imaging requires ultrafast lasers with high peak power to excite higher-order optical nonlinearities, which increases the risk of photodamage and limits its applications in biology.

[0005] On the other hand, infrared spectroscopy has high sensitivity due to its large scattering cross-section and low photon energy, resulting in very little photodamage. In recent years, it has attracted increasing attention due to the development of mid-infrared photothermal (MIP) imaging technology. The resolution of conventional infrared imaging is limited to a few micrometers due to the diffraction limit, which is far from the resolution of a typical optical microscope. Near-field infrared imaging technology, for example, atomic force microscope-based infrared spectroscopy (AFM-IR), has achieved nanoscale spatial resolution, but the use of physical probes limits its wide range of applications, especially intracellular imaging. MIP, on the other hand, integrates molecular vibrational spectroscopy into photothermal imaging technology, enabling three-dimensional imaging of living cells and distant-field coupled selection in organisms. The resolution of MIP imaging reaches the 300 nm level, and by using short-wavelength visible light as detection light and detecting the photothermal effect caused by molecular vibrational absorption, it surpasses the diffraction limit of conventional infrared imaging by an order of magnitude. MIP imaging meets the needs of long-range, non-contact, and non-invasive infrared imaging techniques, but it is an imaging method limited by the diffraction limit of the detected light, and its resolution is far from meeting the requirements of super-resolution. Therefore, there is currently no universally applicable label-free super-resolution imaging method in this field. [Overview of the project] [Problems that the invention aims to solve]

[0006] In view of the above, the object of the present invention is to provide a non-fluorescence super-resolution imaging method based on photothermal relaxation localization microscopy, which enables label-free, highly sensitive, wide spectral range, and low photodamage far-field super-resolution imaging, and makes it possible to achieve a spatial resolution of approximately 100 nm in super-resolution imaging.

[0007] To achieve the above objectives of the present invention, an embodiment provides a non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope, comprising a pump light source, a detection light source, a microscope, a photodetector, a multi-harmonic signal extraction device, and an imaging processing device. The stage of the microscope is equipped with a sample to be detected, capable of performing a three-dimensional spatial displacement scan on the sample to be detected, and has at least one objective lens that focuses the laser beam so that the focus of the pump light and the detection light overlap. The pump light source provides pulsed pump light, which is focused onto the sample to be detected by the objective lens, and the sample to be detected undergoes selective linear absorption of the pump light, dissipating energy through photothermal relaxation, thereby producing a photothermal lens effect. The aforementioned detection light source provides continuous detection light, which is focused by an objective lens and irradiates the sample to be detected on which the pump light acts. This action causes the detection light to change spatially and temporally, i.e., to be modulated. This modulation allows for the detection of smaller spatial structures, mainly in the frequency domain, at higher harmonic frequencies, and exhibits the characteristic of exceeding the diffraction-limited resolution of the detection light. The aforementioned photoelectric detector collects modulated detection light, converts it into an electrical signal, and inputs it to a multi-harmonic signal extraction device. The aforementioned multi-harmonic signal extraction device mixes an electrical signal with a sine wave signal of a harmonic frequency output from a harmonic generator, and then extracts the multi-harmonic signal by low-pass filtering. The imaging apparatus provides a non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope, in which super-resolution imaging is performed based on multiple harmonic signals extracted during three-dimensional spatial displacement scanning, each order of the harmonic signal forms an image, the higher the order, the higher the resolution of the formed image, and images with progressively increasing resolution constitute an image sequence.

[0008] Preferably, the super-resolution imaging system includes a first detection optical path, and based on the first detection optical path, a first detection mode is realized, in which the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves backward detection with respect to the sample to be detected. The first detection optical path involves pump light output from a pump light source passing through a second objective lens and being focused on the sample to be detected; detection light output from a detection light source passing through a beam splitter and being input to the first objective lens, being focused by the first objective lens to illuminate the sample on which the pump light acts; detection light modulated by the sample to be detected being reflected by the beam splitter, being focused by a lens after being reflected by the beam splitter, passing through a pinhole, being filtered by a filter and received by a photodetector.

[0009] Preferably, the super-resolution imaging system includes a second detection optical path, and a second detection mode is realized based on the second detection optical path, in which the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves forward detection with respect to the sample to be detected. In the second detection light path, the pump light output from the pump light source passes through a dichroic mirror and is input to the second objective lens, where it is focused onto the sample to be detected. The detection light output from the detection light source is focused through the first objective lens and illuminates the sample on which the pump light acts. The detection light, modulated by the sample to be detected, passes through the sample to be detected, is input to the dichroic mirror through the second objective lens, is reflected by the dichroic mirror, is focused by a lens, passes through a pinhole, is filtered by a filter, and is received by a photodetector.

[0010] Preferably, the super-resolution imaging system includes a third detection optical path, and a third detection mode is realized based on the third detection optical path, in which the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves backward detection with respect to the sample to be detected. In the third detection light path, pump light output from the pump light source is reflected by a dichroic mirror and input to the first objective lens, where it is focused on the sample to be detected. Detection light output from the detection light source passes through a beam splitter, then through a dichroic mirror and input to the first objective lens, where it is focused and irradiates the sample to be detected on which the pump light acts. The detection light, modulated by the sample to be detected, is reflected by the first objective lens and returns to the dichroic mirror, passes through the dichroic mirror, is reflected by a beam splitter, focused by a lens, passes through a pinhole, is filtered by a filter, and is received by a photodetector.

[0011] Preferably, the super-resolution imaging system includes a fourth detection optical path, and based on the fourth detection optical path, a fourth detection mode is realized, in which the detection light from the detection light source and the pump light from the pump light source are in the same direction within the objective lens, and the detection light achieves forward detection with respect to the sample to be detected. In the fourth detection light path, pump light output from the pump light source is reflected by a dichroic mirror and input to the first objective lens, which focuses it on the sample to be detected. Detection light output from the detection light source passes through the dichroic mirror and input to the first objective lens, which focuses it and illuminates the sample to be detected on which the pump light acts. The detection light modulated by the sample to be detected passes through the sample to be detected, passes through the second objective lens, is focused by the lens, passes through a pinhole and input to a filter, is filtered, and is then received by a photodetector.

[0012] Preferably, the detection light output from the detection light source is first filtered by a single-mode optical fiber and then transmitted through the optical path to the first objective lens.

[0013] Preferably, the detection light source emits continuous detection light consisting of ultraviolet and visible light.

[0014] Preferably, the multi-harmonic signal extraction device employs a multi-channel digital lock-in amplifier, which performs multi-harmonic demodulation on the input electrical signal and extracts the multi-harmonic signal in the frequency domain. When performing multi-harmonic demodulation of an input electrical signal using a multi-channel digital lock-in amplifier, the fundamental frequency used is the pulse repetition frequency of the pump light source.

[0015] Preferably, in the case of mid-infrared light, the objective lens is a reflective objective lens, including Cassegrain objective lenses and Schwarzschild objective lenses, and the reflective objective lens is used to optimize the focusing of pump light in the mid-infrared band.

[0016] Preferably, the objective lens employs a high numerical aperture objective lens such as an air lens, water immersion lens, or oil immersion lens to optimize the focusing of the detected light.

[0017] Compared to conventional technology, the beneficial effects of the present invention include at least the following: (1) After demodulating the detection light, which is modulated by the photothermal effect and dissipation of photothermal energy of the sample to be detected, using multiple harmonics, super-resolution imaging is performed based on the higher-harmonic signals extracted from the frequency domain. This enables super-resolution imaging of light-absorbing molecules or structures without fluorescent labeling, fundamentally solving several limitations of conventional super-resolution imaging, such as photobleaching, labeling efficiency, and labeling selectivity of fluorescent molecules on which it relies. Furthermore, it eliminates, in principle, various limitations of super-resolution fluorescence, such as specially designed fluorescent molecules and photodamage by high-power lasers. (ii) Compared to conventional diffraction-limited photothermal imaging, the present invention further breaks through the resolution limit of detected light and has superior resolution compared to conventional photothermal imaging. Lateral and axial resolution have also been greatly improved. In particular, in photothermal high-harmonic extraction imaging under mid-infrared pumping, it is possible to detect and distinguish smaller lipid droplets and protein structures within cells. (iii) The pump optical power dependence of the present invention is linear under photothermal high harmonics, avoiding the extremely high pump optical energy and photodamage to the sample that are required for conventional nonlinear photothermal excitation. (iv) The present invention has broad compatibility. The present invention can be applied to obtain super-resolution capabilities based on a general photothermal imaging microscope and can simultaneously output image sequences from the fundamental frequency to higher harmonics. Furthermore, the present invention is compatible by design with fluorescence imaging and nonlinear photothermal imaging. (5) The present invention includes, but is not limited to, electron absorption, vibration absorption, and vibration-rotation absorption, and is not limited to the absorption of light involved, and has practical applications in many fields such as materials science, biology, and medicine. [Brief explanation of the drawing]

[0018] To more clearly illustrate embodiments of the present invention or technical solutions in the prior art, the drawings that may be used in describing the embodiments or prior art are briefly introduced below. Clearly, the drawings in the following description represent only a few embodiments of the present invention, and those skilled in the art can obtain other drawings based on these without any creative effort.

[0019] [Figure 1]A diagram showing the principle of photoacoustic relaxation imaging provided by an embodiment. (a) is an energy level diagram and temperature response of the photoacoustic process. (b) is a photoacoustic (PT) process excited by a pulse sequence, and the sample switches between two states of heating (I) and photoacoustic dissipation (II). (c) is a photoacoustic signal detected at the center and edge of an object, showing different relaxation characteristics due to position-dependent photoacoustic dissipation, that is, there is a more significant expansion effect at the edge, doing work outward, and as a result, the temperature rise at that position is lower. (d) is the harmonic component detected at the center and edge of (c), and H1, H2,..., Hn are the demodulated harmonic orders. (e) is the simulation result of the first harmonic temperature response of two 200 nm microspheres separated by 40 nm. (f) is the simulation result of the ninth harmonic temperature response of two 200 nm microspheres separated by 40 nm, and the scale bar is 200 nm. (g) is the process of temperature change at the center and edge of the microsphere. (h) is the intensity ratio of the intermediate signal and the edge signal in the first to ninth harmonics. [Figure 2] It is a structural diagram of a non-fluorescent molecule super-resolution imaging system provided by this embodiment. [Figure 3] It is a schematic diagram of extracting a multiple-order harmonic signal by a multiple-order harmonic signal extraction device provided by this embodiment. [Figure 4] It is a schematic diagram of four detection modes provided by this embodiment: (a) rear detection, counter-pumping; (b) front detection, counter-pumping; (c) rear detection, co-directional pumping; (d) front detection, co-directional pumping. [Figure 5]This figure shows the E-PEARL imaging performance characteristics and cell imaging applications provided by this embodiment. Here, (a) is a TEM image of 30 nm synthetic gold nanoparticles (AuNPs) with a scale bar of 100 nm. (b) is a conventional photothermal imaging (H1) of gold nanoparticles. (c) is E-PEARL imaging of a single 30 nm gold nanoparticle at different harmonic orders. (d) is axial E-PEARL imaging at the 1st, 4th, 7th, and 11th harmonics with a scale bar of 500 nm. (e) shows the lateral resolution (dots) and fitting results (dashed lines) of PEARL imaging at different harmonic orders, with error bars representing the standard deviation of the 2D Gaussian fitting of the imaging. (f) shows the relationship between the E-PEARL signal, signal-to-noise ratio, and harmonic order of a single gold nanoparticle. (g) shows the pump optical power dependence of the E-PEARL signal of a single 30 nm gold nanoparticle at selected harmonic orders (1, 9, 20). (h) is a reflected light imaging of a cell that takes up gold nanoparticles. (i) is an E-PEARL imaging of gold nanoparticles (AuNPs) in the same cell, with a scale bar of 5 μm. (j) is a magnified image of (i), used for resolution comparison with conventional photothermal imaging, with the left figure being H1 and the right figure being H5, with a scale bar of 5 μm. (k) and (m) are cross-sectional images of the signal intensity indicated by arrows in the H1 and H5 images of (j). [Figure 6]The performance characteristics of V-PEARL imaging under mid-infrared absorption provided by the examples are as follows: (a) is V-PEARL imaging of a 200 nm PMMA microsphere under the 1st to 12th harmonics in the 1730 cm⁻¹C=O band. (b) is the measurement of the photothermal relaxation trace of a 2 μm PMMA microsphere, with the edge at 900 nm from the center. (c) is the FFT of the photothermal relaxation trace of (b). (d) is the harmonic intensity ratio (center / edge) of (c). (e) is the full width at half maximum (FWHM) measured at half the maximum value of (a). Error bars represent the standard deviation of the 2D Gaussian fitting of the imaging, with a scale of 200 nm. (f) is the power dependence of the V-PEARL signal when γ-valerolactone is demodulated with the 1st and 4th harmonics. [Figure 7] The examples provide V-PEARL imaging of mature chondrocytes, where (a) and (b) are V-PEARL imaging of mature chondrocytes in the 1750 cm⁻¹ lipid C=O band and 1650 cm⁻¹ amide I band under 8th harmonic, with dotted circles indicating cell nuclei and arrows indicating protein droplets and cytoskeleton. (c) is a V-PEARL imaging integrating lipid channels (a) and protein channels (b). (d) are photothermal spectra of lipid and protein droplets obtained at the points shown in (a) and (b). (e) is an enlarged image of the region indicated by the dotted frame in (a) using conventional MIP (H1) and V-PEARL (H8), with arrows representing small lipid droplets displayed in the V-PEARL imaging. (f) is a cross-sectional view of the 1st and 8th harmonic demodulated signals indicated by the white arrows in (e), with a scale bar of 10 μm. [Figure 8]V-PEARL imaging of live Saccharomyces cerevisiae cells provided by the examples, where (a) is a reflection image of yeast cells; (b) and (c) are V-PEARL images of yeast cells demodulated with the 4th harmonic of the 1750 cm⁻¹ lipid C=O stretching vibration and the 1650 cm⁻¹ amide I band, respectively; (d) is the size distribution of the measured lipid droplets of (b), demodulated with the 1st and 4th harmonics; (e) is the spatial frequency spectrum of (a), (b), and (c); (f) is a reflection imaging diagram of yeast cells; (g) and (h) are V-PEARL images of yeast cells of the 1750 cm⁻¹ lipid C=O band and the 1650 cm⁻¹ amide I band, respectively; (i) is a V-PEARL superposition image of the white boxes of (b) and (c). (j) Intensity curves of two droplets in yeast cells (indicated by the arrows in (h)) and Gaussian fitting, with the curves vertically offset for visual clarity. (k) Features sub-100 nm observed in yeast cells by V-PEARL. (m) Cross-sectional intensity diagram and Gaussian fitting of (k), with the scale bar in the figure being 1 μm. [Modes for carrying out the invention]

[0020] To further clarify the object, technical solutions, and advantages of the present invention, the invention will be described in more detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are used solely for illustrative purposes and are not intended to limit the scope of protection of the invention.

[0021] Conventional fluorescence super-resolution imaging relies on fluorescent labels, but it suffers from limitations such as the cytotoxicity and low labeling efficiency of fluorescently labeled molecules, and the difficulty of labeling small molecules, thus limiting super-resolution imaging. Furthermore, considering the risks of photodamage due to the high laser power required for label-free super-resolution imaging and the limitations on its application to biological systems, this invention provides a non-fluorescent molecule super-resolution imaging system based on photothermal relaxation localization microscopy. This system eliminates the need for additional fluorescent labels in super-resolution imaging, expanding application scenarios to any light-absorbing molecule or structure, and offering invaluable value in research and application fields such as biology, medicine, materials science, and physics.

[0022] Research has shown that the optical diffraction limit can be broken by optically detecting heterogeneous photothermal relaxation. This technique is called photothermal relaxation localization (PEARL). The concept of PEARL microscopy is the localization of the temporal characteristics of the photothermal relaxation process. The photothermal process generated by pulsed pump photoexcitation involves two stages: a heating stage caused by vibrational absorption and a photothermal energy dissipation stage caused by thermal relaxation, as shown in heating stage I and photothermal dissipation stage II in Figure 1(a). For a homogeneous sample, the energy absorbed by molecules per unit volume is constant, but the final photothermal temperature rise effect is different, because the absorbed energy tends to be converted into photoacoustic components at the edges by expansion, as shown in Figure 1(b-c). This dissipation of photothermal energy proceeds over space and time and is detected by the focus of the detection light, and as shown in Figure 1(d), the high-frequency signal decreases more rapidly at the edges. This characteristic can be extracted in the frequency domain, and there are various extraction methods, one of which is to perform higher-order harmonic demodulation using a lock-in amplifier.

[0023] To further explain the mechanism of PEARL's super-resolution imaging, the examples provide results of PEARL resolution improvement obtained through computational simulation. The sample is modeled using two polymethyl methacrylate (PMMA) microspheres with a diameter of 200 nm and a distance of 40 nm. The higher-order harmonic components of the temperature-varying field are extracted using the Fast Fourier Transform, and the final result shows a significant improvement in resolution, as shown in Figure 1(d). Furthermore, a clear difference is observed in the temporal changes of photothermal relaxation between the center and edges of the microspheres, and as shown in Figure 1(g~h), the ratio between the center and edges also increases in the higher-order harmonics, indicating a clear improvement in resolution.

[0024] Based on the above, the embodiment provides a non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope, as shown in Figure 2, which includes a pump light source 1, a detection light source 2, a microscope 3, a photodetector 4, a multi-harmonic signal extraction device 5, and an imaging processing device 6.

[0025] The stage of the microscope 3 is equipped with a sample to be detected, capable of performing a three-dimensional spatial displacement scan on the sample to be detected, and has at least one objective lens that focuses the laser beam so that the focus of the pump light and the detection light overlap. Pump light source 1 provides pulsed pump light, which is focused onto the sample to be detected by an objective lens. The sample selectively absorbs the pump light, dissipating energy through photothermal relaxation and generating a photothermal lens effect. Detection light source 2 provides continuous detection light, which is focused by an objective lens and irradiates the sample on which the pump light acts. This action causes the detection light to change spatially and temporally, i.e., to be modulated. This modulation allows for the detection of smaller spatial structures, mainly in the frequency domain, at higher harmonic frequencies, and can exceed the diffraction-limited resolution of the detection light. The photodetector 4 collects modulated detection light, converts it into an electrical signal, and inputs it to a multi-harmonic signal extractor. As shown in Figure 3, the multi-harmonic signal extractor 5 mixes the electrical signal with the sine wave signal of the harmonic frequency output from the harmonic generator, extracts the multi-harmonic signal through low-pass filtering, and the imaging processing device 6 performs super-resolution imaging based on the multi-harmonic signal.

[0026] When the sample to be inspected is scanned in three-dimensional spatial displacement using the microscope 1, the photodetector 4 collects the detected light at the scanning position of each pixel in real time and converts it into an electrical signal. The multi-harmonic signal extractor 5 extracts multi-harmonic signals from the electrical signal in real time. These multi-harmonic signals include signals from the fundamental frequency (H1) to the higher harmonic (H25), and even higher harmonic signals. The same-order harmonic signals (voltage values) at all pixel positions of the sample to be inspected are integrated to form an image corresponding to one resolution. The higher the order, the higher the resolution of the formed image, and images with sequentially increasing resolution until the resolution reaches approximately 100 nm form an image sequence.

[0027] As shown in Figure 4, four different detection modes can be achieved by configuring different detection optical paths.

[0028] As shown in Figure 4(a), the super-resolution imaging system includes a first detection optical path, and a first detection mode is realized based on this first detection optical path. In this first detection mode, the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves back detection relative to the sample to be measured. This is abbreviated as back detection or opposing pumping. In the first detection optical path, the pump light output from the pump light source is focused on the sample to be detected through the second objective lens, the detection light output from the detection light source passes through the beam splitter and is input to the first objective lens, is focused by the first objective lens and irradiates the sample to be detected on which the pump light acts, the detection light modulated by the sample to be detected is reflected and returns to the beam splitter, is reflected from the beam splitter, is focused by a lens and passes through a pinhole, is filtered by a filter and received by a photodetector. The adopted pinhole structure achieves spatial filtering by confocalizing the detection light, and improves the quality of the laser spot, especially by improving the resolution along the Z axis.

[0029] As shown in Figure 4(b), the super-resolution imaging system includes a second detection optical path, and based on this second detection optical path, a second detection mode is realized. In this second detection mode, the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves forward detection with respect to the sample to be detected. This is abbreviated as forward detection or opposing pumping. Here, the second detection optical path is such that the pump light output from the pump light source passes through a dichroic mirror and is input to the second objective lens, is focused on the sample to be detected via the second objective lens, the detection light output from the detection light source is focused by the first objective lens and illuminates the sample to be detected on which the pump light acts, the detection light modulated by the sample to be detected passes through the sample to be detected and is input to the dichroic mirror via the second objective lens, is reflected by the dichroic mirror, is focused by a lens and passes through a pinhole, is filtered by a filter and received by a photodetector. Here, the pinhole is used for confocal detection and improves the resolution of the Z axis.

[0030] As shown in Figure 4(c), the super-resolution imaging system includes a third detection optical path, and a third detection mode is realized based on this third detection optical path. In this third detection mode, the detection light from the detection light source and the pump light from the pump light source are in the same direction within the objective lens, and the detection light achieves backward detection with respect to the sample to be detected. This is abbreviated as backward detection or co-directional pumping. The third detection optical path involves pump light output from a pump light source being reflected by a dichroic mirror and input to the first objective lens, which focuses the light onto the sample to be detected. The detection light output from the detection light source passes through a beam splitter, then through a dichroic mirror and input to the first objective lens, where it is focused and irradiates the sample on which the pump light acts. The detection light, modulated by the sample to be detected, is reflected by the first objective lens and returns to the dichroic mirror, passes through the dichroic mirror, is reflected by a beam splitter, focused by a lens, passes through a pinhole, is filtered by a filter, and is received by a photodetector.

[0031] As shown in Figure 4(d), the super-resolution imaging system includes a fourth detection optical path, and a fourth detection mode is realized based on the fourth detection optical path, in which the detection light from the detection light source and the pump light from the pump light source are in the same direction within the objective lens, and the detection light achieves forward detection with respect to the sample to be detected, which is abbreviated as forward detection or co-directional pumping. The fourth detection optical path is such that the pump light output from the pump light source is reflected by a dichroic mirror and input to the first objective lens, and is focused on the sample to be detected by the first objective lens, the detection light output from the detection light source passes through the dichroic mirror and is input to the first objective lens, is focused by the first objective lens and illuminates the sample to be detected on which the pump light acts, the detection light modulated by the sample to be detected passes through the sample to be detected, passes through the second objective lens, is focused by the lens, passes through a pinhole and is input to a filter, is filtered and then received by a photodetector.

[0032] In the embodiment, the photodetector may employ a high-bandwidth photodiode, and the detected light can be collected in the forward or reverse direction via the high-bandwidth photodiode. As shown in Figure 4, a lens group is provided at the output end of the detected light to change the propagation direction. The number and position of the reflective and transmissive mirrors that specifically constitute the lens group are not particularly limited as long as they guide the transmission of the modulated detected light. The detected light output from the detection light source is first filtered by a single-mode optical fiber, and then transmitted through the optical path to the first objective lens, and the image quality of the imaging system can be improved by filtering with the single-mode optical fiber.

[0033] In this embodiment, as shown in Figure 4, a lens group is provided at the input end of the filter to focus the optical path. Specifically, a pinhole is provided between two convex lenses included in the lens group. This pinhole is used for confocal detection of the detected light, eliminating interference from stray light and specular reflection, and improving the resolution along the Z axis.

[0034] As shown in Figures 4(a) and 4(b), in the non-fluorescent molecule super-resolution imaging system provided by this embodiment, the pump light and detection light irradiating the sample to be detected are in opposite directions and lie on the same straight line. This design allows for the individual optimization of two lasers with a large wavelength difference and spatial fine-tuning so that the two focal points overlap, thereby maximizing the photothermal signal. Furthermore, as shown in Figures 4(a) and 4(c), back detection can also be performed depending on the actual situation.

[0035] In the embodiment, the multi-harmonic signal extraction device employs a multi-channel digital lock-in amplifier, which performs multi-harmonic demodulation on the input electrical signal to extract the multi-harmonic signal in the frequency domain. When multi-harmonic demodulation is performed on the input electrical signal using the multi-channel digital lock-in amplifier, the fundamental frequency used is the pulse repetition frequency of the pump light source.

[0036] In the non-fluorescent molecule super-resolution imaging system provided by this embodiment, the pump light source emits pump light with wavelengths ranging from visible light to mid-infrared light, and various molecules can be detected by pump light in this range. In this embodiment, a reflective objective lens such as a Cassegrain objective lens or a Schwarzschild objective lens is used as the objective lens of the microscope for the mid-infrared wavelength pump light, and this objective lens is used to optimize the focusing of the pump light in the mid-infrared wavelength band. For the detection light, a high numerical aperture objective lens may be used as the objective lens of the microscope, and this high numerical aperture objective lens is used to optimize the focusing of the detection light and improve the resolution.

[0037] The imaging applications of the above-mentioned non-fluorescent molecule super-resolution imaging system, along with specific target samples, are described below.

[0038] When visible light pumping is used for photothermal relaxation localization imaging of electron absorption (abbreviated as E-PEARL imaging), a pulsed nanosecond laser with a wavelength of 532 nm is used as the pump light source, and a continuous wave laser with a wavelength of 638 nm is used as the detection light source. As shown in Figure 4(c), the pump light emitted from the pump light source and the detection light emitted from the detection light source are collinearly coupled and transmitted to the first objective lens of the microscope, where the beam is focused and irradiates the sample under inspection on the stage, scanning the sample. The sample under inspection vibrates, absorbing the pump light and producing a photothermal effect, after which the photothermal energy is dissipated by photothermal relaxation, resulting in a photothermal lensing effect. The detection light is focused by the objective lens and irradiates the sample under inspection on which the pump light acts, causing the detection light to change spatially and temporally, i.e., be modulated. This modulation is characterized in that, mainly in the frequency domain, smaller spatial structures can be detected at harmonic frequencies, and the diffraction-limited resolution of the detection light can be exceeded. The time-modulated detection light is collected and converted by a photodetector and then input to a multi-channel digital lock-in amplifier. The multi-channel digital lock-in amplifier performs multi-harmonic demodulation on the input detection light. As shown in Figure 3, the fundamental frequency used during demodulation (i.e., the first harmonic) is the repetition frequency of the pump light source, i.e., the number of pulses emitted per second by the pulsed laser. During demodulation, any harmonic order (n-th harmonic, n=1, 2, ..., n) can be selected and demodulated to extract the amplitude and relative phase of each channel. The detection light is converted into an electrical signal by a photodiode, then acquired through high-speed digital-to-analog conversion, and digital demodulation of any harmonic can be performed. Any number of harmonic signals can be executed losslessly, and super-resolution imaging can be performed using the higher-order harmonic signals to acquire a new, sharper image without affecting the conventional photothermal image of the fundamental frequency.

[0039] In this example, as shown in Figure 5(a), we conduct performance studies and cell applications of E-PEARL imaging for gold nanoparticles (AuNP) with a broad absorption band and low cytotoxicity. As shown in Figures 5(c-e), compared to conventional diffraction-limited photothermal imaging shown in Figure 5(b), the spatial resolution of E-PEARL imaging at higher harmonics is significantly improved, exceeding the diffraction limit in both the lateral and axial directions. The resolution improved from 370 nm to 270 nm, and the lateral resolution improved by 1.4 times, exceeding the diffraction limit of the detected light. Due to bandwidth limitations in both the photodetector and the multi-channel digital lock-in amplifier, signals with a harmonic order higher than 22 (11 MHz) were not collected. If the bandwidth of the photodetector and multi-channel digital lock-in amplifier is sufficient, it is possible to collect harmonic signals higher than the 22nd order. Adding a series of new images not only improves the resolution at higher harmonic orders but also offers the possibility of further improving image quality through multi-dimensional image processing.

[0040] As shown in Figure 5(f), the harmonic signals of E-PEARL imaging exhibit a high linear dependence on pump power, indicating that PEARL has a unique mechanism, unlike super-resolution imaging modes based on nonlinear signal generation. Nonlinear signals are generated by higher-order thermal perturbations such as saturation and nonlinear resonance, or nonlinear absorption, which occur under excitation of extremely high pump optical power. Therefore, these modes require strictly sinusoidal pump light, and undesirable effects occur due to arbitrary parasitic frequency components. In contrast, the PEARL signal is generated from the harmonic signals of the relaxation curve, and there should be a linear correlation with the intensity of the pump light. This relationship avoids the use of high-power laser pulses and allows application to a wider range of absorption scenarios.

[0041] In the examples, as shown in Figures 5(h) to (i), the application of E-PEARL imaging to biological systems was demonstrated by imaging the uptake of AuNPs by cancer cells. The uptake of nanostructures into cells has been widely used in photothermal imaging for biological and medical applications such as disease markers. Compared to unmodulated reflection imaging, photothermal imaging has the advantage of a high photothermal conversion rate of AuNPs, and photothermal imaging can obtain high-contrast distribution of AuNPs within cells. Detailed comparisons in Figures 5(j), (k), and (m) show that E-PEARL imaging has superior spatial resolution compared to conventional photothermal imaging and provides a novel method for imaging the cellular distribution and materials of nanostructures.

[0042] Using mid-infrared pumping and visible light detection for localized imaging of photothermal relaxation establishes a PEARL microscope for vibrational spectroscopy, abbreviated as V-PEARL imaging. The 405 nm wavelength detection light emitted from the detection source balances diffraction-limited resolution with photonic damage due to high photon energy. The shorter wavelength increases scattering, increasing the number of photons in the back-detection mode and further improving the signal-to-noise ratio. Cassegrain and high numerical aperture objectives are used to optimize the focusing of the pump beam and probe beam, respectively, and propagate them in opposite directions. The photothermal-modulated detection beam can be collected in either the forward or back-detection mode of a high-bandwidth photodiode. Here, a pinhole is confocally positioned in the back-detection path to eliminate interference from stray light and specular reflection.

[0043] Furthermore, as shown in Figure 6(a), V-PEARL imaging was performed on polymethyl methacrylate (PMMA) microspheres with an average size of 200 nm to measure the resolution, and the infrared pump wavelength was set to 1730 cm⁻¹. -1The C=O stretching vibration peak was set. The full width at half maximum (FWHM) of the PMMA microsphere in the reflected image was approximately 310 nm, but the FWHM measured in the V-PEARL image decreased from 325 ± 3 nm to 126 ± 6 nm at the 10th harmonic, as shown in Figure 6(e). At higher harmonics, the signal-to-noise ratio decreased by approximately 2.8 times, but the resolution improved by 2.6 times. However, considering the high signal level due to infrared absorption and the reduction of 1 / f noise at higher harmonics, this trade-off is experimentally worthwhile for the improvement of resolution.

[0044] In the example, as shown in Figures 6(b) to 6(d), the photothermal relaxation of the detection beam at the center and edge of a single 2 μm PMMA microsphere was also measured. The relaxation at the two locations consisted of one heating process and one dissipation process, namely, one being a fast dissipation on a timescale of tens of nanoseconds and the other a long-duration dissipation on a microsecond scale. The fast dissipation portion was mainly due to photoacoustic transfer, with an intensity decrease of 16% at the center of the microsphere and a decrease of 68% at the edge, indicating a difference in photothermal conversion efficiency. The other portion was a slower exponential decay relaxation, with relaxation times of 3.8 μs and 6.7 μs at the center and edge, respectively. The photothermal relaxation can be described as a Fourier series of various harmonic components shown in the FFT waveform, as shown in Figure 6(c), and the intensity ratio between the center and edge increased at higher harmonics, as shown in Figure 6(d). This is consistent with the simulation results. Furthermore, as shown in Figure 6(f), the linear power dependence of the V-PEARL signal was verified in the example.

[0045] The examples also demonstrate the application of V-PEARL to mammalian cell imaging. While the physiological function of organelles is directly controlled by their spatial distribution, organelle distribution is finely regulated by transport via the cytoskeleton. Lipid droplets (LDs) are emerging as important organelles that play a crucial role in cell survival and state. Multifunctional organelles function as energy storage units, storage spaces for toxic metabolites, or signaling platforms, and their distribution is key to determining their function. Fluorescence-based super-resolution imaging greatly contributes to elucidating the mechanisms of organelle transport and interaction. However, not all biomolecules or organelles can be efficiently fluorescently labeled, and they are often significantly affected by labeled molecules. Here, the examples demonstrate label-free organelle tracking using super-resolution infrared imaging of mammalian cells. As shown in Figures 7(a)-(c), lipids 1750 cm³ -1 C=O symmetric vibrational peak and protein at 1650 cm² -1 V-PEARL imaging was performed on mature chondrocytes cultured for 3 days with an amide I band peak, and the demodulation wavenumber was 8th order. In the example, as shown in Figures 7(a) and (c), a large amount of LD was found to have accumulated within the cell body. Most of the protein signals were distributed in the cytoplasm and cell nucleus, indicating that the chondrocytes were spindle-shaped. As shown in Figure 7(b), several protein droplets were also observed. In the example, as shown in Figure 7(c), it was also confirmed that the LD was densely localized along the cytoskeleton, indicating that the LD is transported via these protein-rich structures. As shown in Figure 7(d), the molecular structures of the LD and protein droplets were verified by in-situ infrared photothermal spectroscopy. Importantly, as shown in Figures 7(e) and (f), V-PEARL can resolve more small LD features in the 300-400 nm range, which is not possible with conventional MIP imaging (MIP, H1) due to insufficient resolution. This holds great potential for the spatial and spectral visualization of organelles, where labeling and molecular information acquisition are difficult.

[0046] This embodiment further provides V-PEARL super-resolution imaging of yeast cells. Yeast is a powerful model organism for studying cell biology, particularly lipid metabolism. However, given that a single yeast cell is approximately 2–4 μm in size, the lack of tools to visualize small LDs (typically 0.05–0.5 μm) is a major limitation. In this embodiment, intracellular far-infrared imaging of yeast cells was performed for the first time using a V-PEARL microscope. As shown in Figure 8(a), the yeast cells contained sparse LDs that were not apparent in the reflected image. Fourth-harmonic V-PEARL imaging, as shown in Figures 8(b) and (c), at 1750 cm⁻¹ -1 A single LD can be seen, 1650cm -1 Protein signals were detected in the amide I band. In the examples, the size distribution of intracellular LDs was also measured, as shown in Figure 8(d), and a clear difference was shown in the histograms of LD sizes for first-harmonic demodulation and fourth-harmonic demodulation. The average size of these LDs was 197±19 nm for first-harmonic demodulation (conventional MIP) and 185±16 nm for fourth-harmonic demodulation. Furthermore, comparing the spatial frequency distribution of the imaging images, as shown in Figure 8(e), V-PEARL showed richer spatial frequency components, especially at higher spatial frequencies, which represented higher spatial resolution. In addition, V-PEARL imaging revealed a subtle spatial separation structure between small LDs and a pair of protein droplets (each approximately 170 nm), as shown in Figure 8(i). Theoretical infrared resolution: 1650 cm⁻¹ -1 Compared to the previous method, this method showed a 43.5-fold improvement in resolution. Simultaneously, line profiles and Gaussian fitting showed a tendency towards improved resolution of the second and third harmonics. In contrast, conventional MIP showed only a single peak with a slight shoulder, as shown in Figure 8(j). Furthermore, as shown in Figures 8(k) and (m), the smallest feature observed in yeast V-PEARL imaging was ~86 nm, and this was the first time that features smaller than 100 nm were resolved using far-field infrared imaging.

[0047] While the specific embodiments described above have explained in detail the technical solutions and beneficial effects of the present invention, these are merely the most preferred embodiments and do not limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A non-fluorescent molecule super-resolution imaging system based on photothermal relaxation localization microscopy, comprising a pump light source, a detection light source, a microscope, a photodetector, a multi-harmonic signal extraction device, and an imaging processing device, The microscope stage is capable of placing the sample to be detected and performing a three-dimensional spatial displacement scan on the sample, and is equipped with at least one objective lens that focuses the laser beam so that the focus of the pump light and the detection light overlap. The pump light source provides pulsed pump light, which is focused onto the sample to be detected by an objective lens, and the sample to be detected undergoes selective linear absorption of the pump light, dissipating energy through photothermal relaxation, and its temporal characteristics are determined during energy dissipation. The aforementioned detection light source provides continuous detection light, which is focused by an objective lens and irradiates the sample to be detected on which the pump light acts. This action causes the detection light to change spatially and temporally, i.e., to be modulated, and this modulation allows for the detection of smaller spatial structures, mainly in the frequency domain, at higher harmonic frequencies, exceeding the diffraction-limited resolution of the detection light. The aforementioned photodetector collects modulated detection light, converts it into an electrical signal, and inputs it to a multi-harmonic signal extraction device. The aforementioned multi-harmonic signal extraction device mixes an electrical signal with a sine wave signal of a harmonic frequency output from a harmonic generator, extracts a multi-harmonic signal through low-pass filtering, and has a linear correlation between the multi-harmonic signal and the pump light intensity. The imaging processing device performs super-resolution imaging based on multiple harmonic signals extracted during three-dimensional spatial displacement scanning, and each order of the harmonic signal forms an image, with the resolution of the formed image increasing as the order increases, and the images with sequentially increasing resolution constitute an image sequence. This is a non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope.

2. The super-resolution imaging system includes a first detection optical path, and based on this first detection optical path, a first detection mode is realized, in which the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves backward detection relative to the sample to be detected. The first detection optical path is characterized in that pump light output from a pump light source passes through a second objective lens and is focused on the sample to be detected, detection light output from a detection light source passes through a beam splitter and is input to the first objective lens, is focused by the first objective lens and irradiates the sample to be detected on which the pump light acts, the detection light modulated by the sample to be detected is reflected by the beam splitter, after being reflected by the beam splitter is focused by a lens and passes through a pinhole, is filtered by a filter and is received by a photodetector, the non-fluorescent molecule super-resolution imaging system according to claim 1.

3. The super-resolution imaging system includes a second detection optical path, and based on this second detection optical path, a second detection mode is realized, in which the detection light from the detection light source and the pump light from the pump light source face each other within the objective lens, and the detection light achieves forward detection with respect to the sample to be detected. The second detection light path is characterized in that pump light output from a pump light source passes through a dichroic mirror and is input to a second objective lens, is focused on the sample to be detected after passing through the second objective lens, detection light output from a detection light source is focused through a first objective lens and irradiates the sample to be detected on which the pump light acts, detection light modulated by the sample to be detected passes through the sample to be detected, is input to a dichroic mirror after passing through the second objective lens, is reflected by the dichroic mirror, is focused by a lens, passes through a pinhole, is filtered by a filter and received by a photodetector, the non-fluorescent molecule super-resolution imaging system according to claim 1.

4. The super-resolution imaging system includes a third detection optical path, and based on this third detection optical path, a third detection mode is realized, in which the detection light from the detection light source and the pump light from the pump light source are in the same direction within the objective lens, and the detection light achieves backward detection with respect to the sample to be detected. The third detection light path is characterized in that pump light output from a pump light source is reflected by a dichroic mirror and input to a first objective lens, the first objective lens focuses on the sample to be detected, detection light output from a detection light source passes through a beam splitter, passes through a dichroic mirror and input to the first objective lens, is focused by the first objective lens and irradiates the sample to be detected on which the pump light acts, the detection light modulated by the sample to be detected is reflected by the first objective lens and returns to the dichroic mirror, passes through the dichroic mirror, is reflected by a beam splitter, is focused by a lens and passes through a pinhole, is filtered by a filter and received by a photodetector, the non-fluorescent molecule super-resolution imaging system according to claim 1.

5. The super-resolution imaging system includes a fourth detection optical path, and based on this fourth detection optical path, a fourth detection mode is realized, in which the detection light from the detection light source and the pump light from the pump light source are in the same direction within the objective lens, and the detection light achieves forward detection with respect to the sample to be detected. The fourth detection light path is characterized in that pump light output from a pump light source is reflected by a dichroic mirror and input to a first objective lens, which focuses it on the sample to be detected, detection light output from a detection light source passes through a dichroic mirror and input to the first objective lens, which focuses it on the sample to be detected on which the pump light acts, and the detection light modulated by the sample to be detected passes through the sample to be detected, passes through a second objective lens, is focused by a lens, passes through a pinhole and input to a filter, is filtered and then received by a photodetector, the non-fluorescent molecule super-resolution imaging system according to claim 1.

6. A non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope according to claim 2, 3, 4, or 5, characterized in that the detection light output from the detection light source is first filtered by a single-mode optical fiber and then transmitted through the optical path to a first objective lens.

7. The non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope according to claim 1, characterized in that the detection light source emits continuous detection light of ultraviolet and visible light.

8. The aforementioned multi-harmonic signal extraction device employs a multi-channel digital lock-in amplifier, which performs multi-harmonic demodulation on the input electrical signal and extracts the multi-harmonic signal in the frequency domain. A non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope according to claim 1, characterized in that when performing multiple-harmonic demodulation of an input electrical signal using a multi-channel digital lock-in amplifier, the fundamental frequency used is the pulse repetition frequency of the pump light source.

9. A non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope according to claim 1, characterized in that, in the case of mid-infrared light, the objective lens employs a reflective objective lens including a Cassegrain objective lens and a Schwarzschild objective lens, and the reflective objective lens is used to optimize the focusing of pump light in the mid-infrared band.

10. A non-fluorescent molecule super-resolution imaging system based on a photothermal relaxation localization microscope according to claim 1, characterized in that the objective lens employs a high numerical aperture objective lens such as an air lens, a water immersion lens, and an oil immersion lens to optimize the focusing of detected light.

Images