Stimulated Raman photo-thermal microscope

Abstract

A stimulated Raman photothermal (SRP) microscope for imaging a sample. The first light source omits an intensity modulated pump beam. The second light source omits an intensity modulated Stokes beam. The Stokes beam is combined with the pump beam to form a composite beam. The composite beam is directed to a sample to induce thermal effects caused by the stimulated Raman process. A third light source emits a probe beam that is directed to the sample. And after the detection light beam is modulated by the sample, the optical detector detects the modulation condition of the detection light beam so as to measure the stimulated Raman photo-thermal signal.

Description

[0001] Government funding

[0002] This invention was made with U.S. government support under Grant No. R35GM136223 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 441,297, filed on January 26, 2023, entitled “Stimulated Raman Photothermal (SRP) Microscopy,” the entire contents of which are incorporated herein by reference as if fully set forth herein. Technical Field

[0005] The present disclosure relates to microscopy, and more particularly, to stimulated Raman photothermal (SRP) microscopy. Background Art

[0006] Stimulated Raman scattering (SRS) microscopy has developed into one of the most powerful tools in the field of label-free imaging. In stimulated Raman scattering, two temporally and spatially coincident laser pulse trains (e.g. Figure 1A102 and 104 in the laser field, namely the pump and Stokes fields, coincide in space and time. They coherently act on Raman-active molecules resonating with the laser's beat frequency, resulting in stimulated Raman gain (SRG) in the Stokes field and stimulated Raman loss (SRL) in the pump field. Stimulated Raman scattering provides the same molecular vibrational signatures as traditional Raman spectroscopy, but with acquisition speeds up to six orders of magnitude higher. This speed increase, combined with the development of vibrational probes, has enabled a wide range of applications. These include label-free stimulated Raman histology, nutrient mapping, the study of altered cancer metabolism, the resolution of heterogeneity in the microbiome, and the rapid detection of antimicrobial susceptibility. However, despite these advances, stimulated Raman scattering still faces inherent challenges in detection sensitivity due to low modulation depth (less than 0.1% for pure liquids) and shot noise in the pump or Stokes beam. Simply increasing the number of photons can easily exceed the power tolerance limit of the sample.

[0007] In order to push the fundamental limit of stimulated Raman scattering sensitivity, it is necessary to reduce the measurement noise or amplify the signal. To reduce the measurement noise, researchers have focused on using squeezed light, also known as "quantum-enhanced stimulated Raman scattering (quantum-enhanced SRS)". Studies have shown that the use of continuous wave squeezed light can achieve a signal-to-noise ratio (SNR) enhancement of 3.6 dB, and the use of pulsed compressed light can achieve a SNR enhancement of 2.89 dB, without additional interference to the sample. This method is promising, but it is also limited by low compression efficiency and decoherence in complex imaging systems. In order to enhance the signal, a variety of photophysical processes are used to increase the cross section. These processes include electronic pre-resonance stimulated Raman scattering (electronic pre-resonance SRS), plasmon-enhanced SRS, and stimulated Raman excited fluorescence (stimulated Raman excited fluorescence). These methods have achieved extremely high stimulated Raman scattering signal enhancement factors (10 4 to 10 7 ) and achieve single-molecule stimulated Raman scattering measurements. However, its reliance on specific target molecules or plasmonic nanostructures limits its application.

[0008] In order to explore the path to enhance the signal, the physical mechanism of stimulated Raman scattering is re-examined from the perspective of energy transfer from the laser field to the sample. Figure 1B As shown in Figure 1, when pump pulses and Stokes pulses with appropriate wavelengths (109 and 111, respectively) interact with Raman-active molecules, the target molecules are excited to their vibrationally excited states. The energy required for this transition is exactly equal to the beat frequency between the pump laser and the Stokes laser (see also Figure 6A Importantly, following stimulated Raman scattering excitation, the vibrationally excited molecules rapidly release their vibrational energy through non-radiative decay (see step 606 of method 600 ). This, in turn, heats the surrounding environment, inducing a stimulated Raman photothermal (SRP) effect (step 608 of method 600 ). Previous studies have shown that, from the perspective of photodamage in coherent anti-Stokes Raman scattering (CARS) imaging, approximately 0.08% of the laser power is absorbed by myelin samples through simultaneous stimulated Raman gain and loss processes.

[0009] Photothermal microscopy with optical detection has been fully developed, achieving sensitivity down to the single-molecule level. In photothermal spectroscopy, which dates back to the 1970s, optical absorption raises the local temperature and induces a localized change in the refractive index, which is then measured by a probing beam. Early photothermal microscopy techniques focused primarily on electronic absorption, targeting non-fluorescent dye molecules or metallic nanostructures. More recently, mid-infrared photothermal (MIP) microscopy offers versatile infrared-active vibrational spectroscopy. This technique offers micromolar sensitivity with spatial resolution approaching the diffraction limit of visible light. Furthermore, even higher resolution can be achieved by detecting high harmonic signals. In contrast, thermal effects induced by the Raman process are generally considered minimal due to the small cross section of Raman scattering.

[0010] In addition, fiber lasers have the advantages of strong environmental adaptability and fast wavelength tunability, which are crucial in the translational application of microscopy systems. Using a rapidly tunable dual-output all-fiber optical parametric oscillator (OPO), it is now possible to use portable fiber lasers to perform stimulated Raman scattering, covering a wide spectral range (700–3100 cm -1 ), where an auto-balance detection system is required to eliminate the high laser noise in the fiber laser. However, the auto-balancing device is complex and susceptible to electronic interference in the environment, which limits the sensitivity of the fiber laser-based stimulated Raman scattering system. Another challenge facing stimulated Raman scattering is the cross-phase modulation background. High numerical aperture objectives are required for signal collection to minimize this background, making the operation unfriendly to non-expert users. Summary of the Invention

[0011] In response to these needs, the present disclosure proposes a system and method for stimulated Raman photothermal (SRP) microscopy. This system and method challenges conventional wisdom by nonlinearly benefiting from high laser peak power for low-duty-cycle coherent Raman excitation. This disclosure demonstrates the significant thermal effects of stimulated Raman scattering (SRS) and demonstrates its potential for ultra-sensitive bond-selective imaging.

[0012] In at least one aspect, the present disclosure proposes a system and method for stimulated Raman photothermal microscopy (SRT), which exhibits superior sensitivity to stimulated Raman scattering (SRS). This system and method achieves imaging by measuring the thermal lensing effect induced by the SRT effect. Simulation results demonstrate that the SRT effect can induce a Kelvin-scale temperature rise at the focal point of SRS, and this simulation result is confirmed by fluorescence thermometer measurements. Based on this, a SRT microscope is constructed by measuring the thermal lensing effect of SRT. In the SRT measurements, a surprisingly high modulation depth (approximately 22.3%) was observed, significantly exceeding the modulation depth of SRS (approximately 0.04%) at the same average laser power. This high modulation depth enables SRT microscopy to achieve exceptionally high sensitivity. This SRT technique has also been demonstrated to be widely applicable to biological imaging at various spatial scales and Raman wavelengths.

[0013] In at least one aspect, the present disclosure provides a stimulated Raman photothermal microscopy system that implements a simplified fiber laser probe beam for stimulated Raman photothermal signals and an air condenser to collect the probe beam. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make it easier for those skilled in the art to understand how to make and use the disclosed system, reference may be made to the following drawings:

[0015] Figure 1A Schematic diagram of stimulated Raman gain and loss.

[0016] Figure 1B Schematic diagram of the stimulated Raman photothermal effect.

[0017] Figure 1C This is a simulation diagram of the temperature rise caused by stimulated Raman photothermal heating in the time domain and space domain.

[0018] Figure 1D This is the simulated distribution of the thermal lens induced by stimulated Raman photothermal heat in pure dimethyl sulfoxide (DMSO).

[0019] Figure 1E Schematic diagram of fluorescence thermometer measurement of temperature rise induced by stimulated Raman photothermal heating.

[0020] Figure 1F This is the fluorescence intensity diagram of rhodamine B in dimethyl sulfoxide during stimulated Raman scattering.

[0021] Figure 2A It is a schematic diagram of the system structure of the stimulated Raman photothermal microscope according to the technical solution disclosed in the present invention.

[0022] Figure 2Byes Figure 2A The image shows the variation of the stimulated Raman photothermal signal measured by the microscope with the modulation duty cycle.

[0023] Figure 2C yes Figure 2A The image shows the variation of the stimulated Raman photothermal signal measured by the microscope with the modulation frequency.

[0024] Figure 2D is used Figure 2A A diagram showing the trajectories obtained by microscopy.

[0025] Figure 3A It is a stimulated Raman photothermal image obtained by a microscope according to the technical solution of the present disclosure.

[0026] Figure 3B This is a stimulated Raman scattering image obtained by a microscope according to the technical solution of the present disclosure.

[0027] Figure 3C These are stimulated Raman photothermal images and stimulated Raman scattering images obtained by the microscope according to the technical solution of the present disclosure.

[0028] Figure 3D It is a Gaussian fitted full width at half maximum (FWHM) diagram of a bead profile obtained by a microscope according to the technical solution of the present disclosure.

[0029] Figure 4A This is a stimulated Raman photothermal image of living SJSA-1 cells in phosphate-buffered saline (PBS) according to the technical solution of the present disclosure.

[0030] Figure 4B yes Figure 4A The image shown is the image after colorization and encoding.

[0031] Figure 4C According to the technical solution disclosed in the present invention, at 2930 cm -1 Hyperspectral stimulated Raman photothermal image of fixed SJSA-1 cells in phosphate-buffered saline.

[0032] Figure 4D yes Figure 4C Phase analysis results of the hyperspectral stimulated Raman photothermal image are shown.

[0033] Figure 4E and Figure 4FThey are images and graphs of stimulated Raman photothermal analysis of SJSA-1 cells treated with palmitic acid-d31 (PA-d31) in phosphate-buffered saline according to the technical solution of the present disclosure.

[0034] Figure 4G and Figure 4H The figures are respectively images and graphs of stimulated Raman photothermal analysis of SJSA-1 cells in phosphate-buffered saline without palmitic acid-d31 (PA-d31) treatment according to the technical solution of the present disclosure.

[0035] Figure 4I –4K is based on the technical solution of this disclosure, at 2930cm -1 Stimulated Raman photothermal images of different areas of mouse brain tissue immersed in phosphate-buffered saline.

[0036] Figure 4L is obtained using a microscope according to the technical solution of the present disclosure, Figure 4I – Representative spectra of mouse brain tissue shown in 4K.

[0037] Figure 5A The microscope was used according to the technical solution of the present invention to obtain the -1 Stimulated Raman thermal image of a single varicella-zoster virus immersed in glycerol-d8.

[0038] Figure 5B It is from Figure 5A The Raman spectrum of a single virus in the CH region is shown.

[0039] Figure 5C The microscope was used according to the technical solution of the present invention to obtain the -1 Stimulated Raman photothermal image of immobilized Mia PaCa-2 cells immersed in glycerol-d8.

[0040] Figure 5D It is from Figure 5C The chemical distribution map derived from phase analysis of the image shown.

[0041] Figure 5E The microscope was used according to the technical solution of the present invention to obtain the -1 Stimulated Raman photothermal spectroscopy image of OVCAR-5 tissue after glycerol-d8 clearing treatment.

[0042] Figure 5F It is from Figure 5E The single-pixel spectral maps of the identified regions of interests (ROI) obtained in .

[0043] Figure 6A Flowchart of thermal effect generation in stimulated Raman photothermal microscopy.

[0044] Figure 6B This is a schematic diagram of a thermal diffusion model of the thermal effect induced by stimulated Raman photothermal heat using the microscope described in the present disclosure, where arrows indicate the direction of heat propagation.

[0045] Figure 7 It is a thermal ball lens model used in a stimulated Raman photothermal microscopy detection scheme according to the technical solution disclosed in the present invention.

[0046] Figure 8 This is a table of properties of various liquid media used in the microscope according to the technical solution of the present disclosure.

[0047] Figure 9 It is a schematic diagram of another embodiment of a stimulated Raman photothermal microscope according to the technical solution of the present disclosure. DETAILED DESCRIPTION

[0048] The present technical solution overcomes many of the difficulties in the prior art associated with stimulated Raman microscopy. The advantages and other features of the systems and methods disclosed herein will become clearer and easier to understand for those of ordinary skill in the art through the detailed description of the representative embodiments described below in conjunction with the accompanying drawings. Similar reference numerals are used herein to refer to similar parts. In addition, directional terms such as "upper", "lower", "distal", and "proximal" used herein are only used to describe the positional relationship between the various components. For example, the "upper" surface only refers to the other side that is different from the "lower" surface of the component, and these terms do not indicate absolute directionality (i.e., the so-called "upper" component is not always located at a higher position in space).

[0049] Reference Figures 1A to 1F , showing the theoretical simulation and experimental observation of the stimulated Raman photothermal effect. More specifically, Figure 1A is a schematic diagram illustrating an exemplary pump pulse 102 and Stokes pulse 104 , as well as stimulated Raman gain (SRG) 106 and loss (SRL) 108 . Figure 1B Schematic diagram of the stimulated Raman photothermal effect. Figure 1C Simulations of the temperature rise induced by stimulated Raman photothermal heating in the time domain (Figure 120) and spatial domain (Image 122) are shown. The spatial scale is 1 micrometer (µm). Figure 1D This is the simulated distribution of the thermal lens induced by stimulated Raman photothermal heating in pure dimethyl sulfoxide (128). Figure 1ESchematic diagram of fluorescence thermometer measurement of temperature rise induced by stimulated Raman photothermal heating with stimulated Raman scattering (SRS) turned off (130) and turned on (132). Figure 1F 140 is a graph of the fluorescence intensity of Rhodamine B in dimethyl sulfoxide during stimulated Raman scattering. Under resonance conditions, the beat frequency (ω p – ω s ) is adjusted to 2931 cm -1 For off-resonance conditions, i.e. background (BG), the beat frequency was adjusted to 2850 cm -1 The (background removed) resonance signal curve 142 is obtained by subtracting the non-resonance curve (background, curve 141) from the resonance curve (dash-dotted curve 144) to eliminate non-photothermal contributions.

[0050] In the simulated stimulated Raman loss measurement, the modulation depth of the pump beam ( ), pump laser intensity (I p ) and the change in the number of photons per pulse ( ) can be expressed as:

[0051]

[0052] in, is the reduced Plank's constant, is the angular frequency of the pump field, is the laser repetition rate. The factor 2 is used to take into account the effect of 50% duty cycle. On this basis, the formula Estimate the energy deposition per stimulated Raman scattering pulse pair, where The literature shows that under the conditions of a Stokes beam of 25 mW (modulated at 50% duty cycle), a pump beam of 15 mW, and a repetition rate of 80 MHz on the sample, the Raman shift of dimethylsulfoxide (DMSO) at 2913 cm -1 The stimulated Raman scattering modulation depth of the Raman mode reached 0.04%. Substituting these measured values ​​into a formula, the energy deposition per laser pulse pair was calculated to be 8.7 femtojoules (fJ), equivalent to 0.7 microwatts (µW) at a pulse interval of 12.5 nanoseconds (ns). This significant energy deposition is consistent with Min's calculation of the stimulated Raman scattering apparent cross section.

[0053] Based on the energy deposition estimate, Fourier's law is applied and a finite element model 630 ( Figure 6B ) to quantitatively simulate the temperature rise induced by stimulated Raman scattering in pure dimethyl sulfoxide. The simulation results (Figure 120) show that, using a 0.8 numerical aperture (NA) objective lens and conventional stimulated Raman scattering laser power (pump laser power of 25 mW, Stokes laser power of 50 mW, repetition rate of 80 MHz, and 50% duty cycle), the temperature rise at the center of the laser focus can reach 2.4 K (plot 121) after 12 microseconds (µs) of stimulated Raman excitation (equivalent to 960 pump / Stokes pulse pairs). At t = 12 µs, the temperature rise at 1 µm (plot 124) and 2 µm (plot 126) from the focus center is 0.54 K and 0.12 K, respectively. Figure 1C The temperature distribution at different time points is also shown in the graph 120. At t = 12 microseconds, the full width at half maximum (FWHM) of the temperature rise field is calculated to be 1.1 micrometers, indicating a very localized thermal effect.

[0054] The increase in temperature then changes the local refractive index through the thermal-optic effect. For a refractive index of 1.497 and a thermo-optic coefficient of -4.93 × 10 -4 Kelvin -1 For pure DMSO with a dn / dT, the heating effect caused by stimulated Raman excitation leads to a 0.07% refractive index drop at the focus center (t = 12 μs). Figure 1D As shown in Figure 128, this change in refractive index extends nonlinearly to the surrounding area through thermal propagation, leading to the formation of a thermal lens. The emergence of this thermal lens forms the basis of stimulated Raman photothermal microscopy.

[0055] The simulation results were verified by a fluorescence thermometer. The emission intensity of some fluorescent molecules shows temperature dependence. For example, at room temperature, the fluorescence intensity of rhodamine B decreases by about 2% for every 1 Kelvin increase in temperature. This property has been used in fluorescence-detected mid-infrared photothermal spectroscopy. This method is used to quantify the temperature rise at the focus of stimulated Raman scattering, and rhodamine B is used as a fluorescence thermometer. When the broadened (chirped) pump laser and the Stokes laser are focused on a dimethyl sulfoxide solution containing rhodamine B, the rhodamine B molecules at the focus of stimulated Raman scattering are electronically excited through a multiphoton absorption process and emit fluorescence. At the same time, when the beat frequency between the pump laser and the Stokes laser is tuned to resonate with the C–H vibration in dimethyl sulfoxide, the stimulated Raman photothermal effect causes the temperature to rise, thereby reducing the two-photon fluorescence intensity of the rhodamine B molecules (compare Figure 1E The experiment used the same parameters as the simulation. First, the fluorescence intensity curve measured at the off-resonance frequency was subtracted from the measurement at the resonant frequency to remove the contribution of photobleaching from the result. It was then observed that after 12 microseconds of resonant stimulated Raman scattering, the fluorescence intensity decreased by about 2.3%, corresponding to a temperature rise of about 1.2 Kelvin ( Figure 1F The temperature rise of the laser is measured in the middle graph (line 144), which is close to the simulated value of 2.4 Kelvin. The difference between the two values ​​can be attributed primarily to their different sampling areas. The experimental measurement shows the average temperature rise across the entire laser focus, while the simulation results only show the temperature rise at the center of the laser focus (approximately 20 nanometers in diameter). After taking a weighted average of the temperature rise across the entire heated area and accounting for the two-photon excitation intensity of the Gaussian beam, the fluorescence curve (line 144) is highly consistent with the simulation result (line 148).

[0056] To rule out the potential influence of thermophoresis on the observed decrease in fluorescence intensity, a control experiment was conducted using Rhodamine 800 (Rh800) as a dye dissolved in dimethyl sulfoxide under the same stimulated Raman scattering and two-photon excitation conditions. The fluorescence intensity of Rh800 does not change with temperature and is not susceptible to photobleaching. The stimulated Raman scattering process did not alter the fluorescence intensity of Rh800, indicating that the influence of thermophoresis is negligible.

[0057] The stimulated Raman photothermal effect can be sensed optically. The stimulated Raman photothermal effect produces a diverging lens and can be detected optically using a continuous wave beam. Figure 7, a hot ball lens model 700 is constructed to illustrate the detection method. The solid line 702 represents the propagation of the detection laser in the absence of a spherical lens, and the dotted line 704 represents the propagation of the detection laser in the presence of a spherical lens. Δn represents the refractive index change caused by the stimulated Raman photothermal effect. In the model 700, it is assumed that the stimulated Raman scattering excitation produces uniform heating in a spherically symmetric region. The hot ball lens changes the propagation of subsequent detection light, which can be expressed in a formula based on geometric optics. Under the paraxial approximation, the model 700 gives the stimulated Raman photothermal signal intensity ( ) is as follows:

[0058] (2)

[0059] In this model, stimulated Raman scattering is modeled as a two-photon vibrational excitation process. The first term ( ) represents the number of stimulated Raman scattering events, that is, the number of vibrational excitations. is the number of molecules in the excitation volume, is the stimulated Raman scattering cross section (unit: square centimeter fourth power·second / photon, cm 4 s / photon), and They are the photon flux of the pump laser and the Stokes laser (unit: photons per square centimeter per second, photons cm -2 ·s -1 ), is the laser pulse width (in seconds). The second term ( ) represents the energy of the vibrational transition. The last term describes the nature of the measurement environment, where is the stimulated Raman scattering excitation volume, is the heat capacity, is the refractive index, and dn / dT is the thermo-optic coefficient. The product of the first two terms gives the energy deposition, while the last term gives the conversion rate of heat to refractive index. This term is inversely proportional to the excitation volume and heat capacity and directly proportional to the thermo-optic coefficient. This environmental property-dependent term retains the potential for signal enhancement, which is not applicable in stimulated Raman scattering measurements. Finite-difference time-domain wave propagation simulations were performed for high numerical aperture (NA) conditions. The results show that the stimulated Raman photothermal signal intensity maintains a linear relationship with concentration.

[0060] Reference Figures 2A to 2D , generally showing an experimental setup 200 of stimulated Raman photothermal microscopy ( Figure 2A), and the characterization results of the stimulated Raman photothermal modulation depth as a function of duty cycle and modulation frequency. Figure 2B ) shows the relationship between the stimulated Raman photothermal signal 242 and the modulation duty cycle. Figure 2C ) shows the relationship between the stimulated Raman photothermal signal 252 and the modulation frequency. Figure 2D ) are shown at 2913 cm -1 and 2850 cm -1 The trajectories obtained under resonant (262) and non-resonant (264) conditions under Raman shift show that stimulated Raman photothermal generation produces a high modulation depth (22.3%) when using dimethyl sulfoxide as the sample.

[0061] Overall, Figure 2A The experimental setup for stimulated Raman photothermal microscopy 200 is shown. Microscope 200 includes a light source, comprising lasers 201a and 201b, which emit a pump beam 202 and a Stokes beam 204. An additional source laser 201c provides a probe beam 208. The system includes dichroic mirrors (DM) 216a and 216b, which direct the laser light and help combine the pump beam 202 and the Stokes beam 204 into a composite pump beam 206. These mirrors also allow for collinear alignment of the composite pump beam 206 with the probe beam 208. Pump beam 202 passes sequentially through a half-wave plate (HWP) 220, a polarizing beam splitter (PBS) 222, an acousto-optic modulator (AOM) 224, and a delay line (DL) 226. The Stokes beam 204 passes through a half-wave plate 220 b, a polarization beam splitter 222 b, and an acousto-optic modulator 224 b in sequence before being combined with the pump beam 202. The composite pump beam 206 and the probe beam 208 are directed to propagate collinearly (i.e., along a common optical path) toward the sample 230 and are scanned by a scanning mirror 228. They then pass through lenses 210 a and 210 b, and an objective lens (OBJ) 229 in sequence before reaching the sample 230.

[0062] Stimulated Raman photothermal microscopy 200 utilizes a third continuous-wave beam 208 to sense local refractive index modulation. Synchronized pump and Stokes pulse trains 202 and 204 are intensity modulated, combined, and stretched by two acousto-optic modulators (AOMs) 204a and 204b. Stretching of the femtosecond pulses produces a spectral focus for excitation of a specific Raman mode. The probe beam 208 is collinearly aligned with the stimulated Raman scattered (SRS) beams 202 and 204 along a shared optical path. A pair of lenses 210a and 210b adjusts the collimation of the probe beam 208, axially offsetting the focus of the probe laser 201c from the SRS focus, thereby maximizing the photothermal signal. An iris 212 at the backfocal plane of the condenser lens (COND) 214 is set to a numerical aperture of 0.4 to convert the probe beam's refractive index modulation into intensity modulation. After the probe beam passes through a spectral filter (SP) 235, its intensity is detected by a photodetector 234 (e.g., a fast photodiode). The detected signal is then passed to a high-pass filter (HP) 236 and a broadband amplifier (AMP) 238. The stimulated Raman photothermal modulation induced by the synchronized pump and Stokes pulses is digitized in real time using a high-speed digitization card.

[0063] It should be understood that other conventional optical components (e.g., lenses, mirrors) and / or electronic components (power supplies, amplifiers, processing devices) may also be included to allow the microscope 200 to operate as described herein. Furthermore, as will be appreciated by those skilled in the art, various components of the microscope 200 may be implemented using components other than those specifically shown and described.

[0064] Unlike stimulated Raman scattering, in stimulated Raman photothermal microscope 200, both pump beam 202 and Stokes beam 204 are intensity modulated. The intensity of stimulated Raman scattering signal is proportional to the product of pump and Stokes peak power. Under the condition of constant average laser power, reducing the laser duty cycle will lead to higher laser peak power, thereby obtaining more stimulated Raman scattering energy deposition. Figure 2BAs shown in graph 240 in , the experimental results confirm this relationship and show a much higher stimulated Raman photothermal signal intensity at a lower duty cycle (graph 242). In stimulated Raman photothermal imaging applications, the duty cycle is set to 5% to 10% to strike a balance between signal intensity and laser power. It is worth noting that matched filtering can be applied to the stimulated Raman photothermal signal with a low duty cycle to further improve the signal-to-noise ratio (SNR). Another key parameter is the modulation frequency. Figure 2C As shown in graph 250 , lower frequencies show higher signal intensity (plot 252 ) due to a longer thermal accumulation time, but are also affected by more l / f laser intensity noise. However, lower frequencies also slow imaging speed and affect spatial resolution. To balance these factors, a frequency of 125 kilohertz (kHz) was selected.

[0065] For pure liquids, at a duty cycle of 5% and a modulation frequency of 125 kHz, the modulation induced on the probe beam 208 is so strong that the stimulated Raman photothermal signal can be measured directly in the DC channel without any amplification ( Figure 2D A reasonable laser power is applied to sample 230 to excite the C–H symmetric stretching vibration mode (2913 cm) in dimethyl sulfoxide. -1 ), the modulation depth reaches 22.3%. This value is more than 500 times higher than the stimulated Raman scattering modulation depth (0.04%) obtained at the same average power. This significantly higher modulation depth lays the foundation for higher detection sensitivity.

[0066] In addition to the duty cycle and modulation frequency, the thermal ball lens model 700 ( Figure 7 ) shows that the properties of the medium are another important factor affecting the intensity of the photothermal signal. Photothermal signal intensity (S), thermo-optic coefficient (dn / dT) and heat capacity ( ) have the following relationship: , which is also supported by existing literature. However, water, the most common medium in biological samples, has a low thermo-optical coefficient (-1.13 × 10 -4 Kelvin -1 , K -1 ) and high heat capacity (4181 joules per kilogram per Kelvin, J·kg -1 ·K -1 In order to improve the signal strength, a variety of common liquid media have been studied, and the results are as follows Figure 8As shown in Table 800 in , the study found that glycerol increased the signal intensity by approximately 3.21 times compared to water. Glycerol also has good biocompatibility and is widely used as a mounting medium or clearing agent in biological imaging. Thermal lens contrast simulations were also consistent with established theory. The simulations showed that under the same heating conditions as those for 100-nanometer polymethyl methacrylate (PMMA) nanoparticles subjected to resonant stimulated Raman photothermal heating, the peak refractive index change in glycerol was approximately 2.5 times that in water. Therefore, glycerol was chosen as the medium to push the sensitivity limit of stimulated Raman photothermal imaging. Given glycerol's inherent Raman-active vibrational characteristics, deuterated glycerol (glycerol-d8) was used for stimulated Raman photothermal measurements in the C–H region and fingerprint region.

[0067] Reference Figures 3A to 3D Characterization of stimulated Raman photothermal spectroscopy and imaging performance is shown. Graphs 310 and 320 show the stimulated Raman photothermal signal and stimulated Raman scattering signal, respectively, of different concentration gradients of dimethyl sulfoxide dissolved in deuterated dimethyl sulfoxide (DMSO-d6). Each graph includes inset graphs 312 and 322, respectively, showing how signal intensity (plots 314 and 324, respectively) varies with concentration. Figure 3C It shows that under the same average laser power and the same field of view, at 2950 cm -1 Stimulated Raman photothermal image 330 and stimulated Raman scattering image 332 of 100 nm polymethyl methacrylate microspheres at 400 nm (scale bar: 500 nm). The microspheres were immersed in deuterated glycerol. Figure 3D is the half-maximum width (FWHM) of the Gaussian fit of the microsphere distribution profile (218 nm).

[0068] First, we used samples with well-defined properties to characterize the spectral fidelity of stimulated Raman photothermal microscopy. The stimulated Raman photothermal spectra were consistent with the stimulated Raman scattering spectra in both bulk liquid samples and nanoparticles (polymethyl methacrylate). The stimulated Raman photothermal intensity was also consistent with the The high spectral fidelity of the stimulated Raman photothermal spectrum lays the foundation for further comparison of the detection sensitivity of the two techniques.

[0069] DMSO was subsequently measured at 2913 cm -1To keep the thermal and optical properties consistent during the measurement, deuterated DMSO (DMSO-d6) was used as a solvent to dilute DMSO. Figure 3A As shown, the stimulated Raman photothermal spectrum of a high-concentration DMSO sample is clear and smooth, with signals visible down to a concentration of 5.1 millimolar per liter (mM). The limit of detection (LOD) of 2.3 mM was calculated using the formula LOD = 3σ / k, where σ is the baseline standard deviation and k is the slope of the intensity-concentration linear calibration curve. In comparison, the limit of detection for stimulated Raman scattering (SRS) is 39 mM at the same average laser power. Therefore, SRSP measurements offer a roughly 17-fold improvement. The limits of detection for C=C bonds and C–D bonds were measured in DMSO using 1,7-octadiyne and deuterated DMSO, respectively. In both cases, SRSP demonstrated superior sensitivity to SRSP, with a 12-fold improvement for 1,7-octadiyne and a 4-fold improvement for deuterated DMSO.

[0070] This increased sensitivity makes high-quality imaging of nanoparticles possible. Using stimulated Raman photothermal microscopy, hyperspectral images of 100-nm polymethyl methacrylate microspheres ( Figure 3C ) was successfully obtained. The stimulated Raman photothermal spectrum obtained was at 2950 cm -1 The Raman peak of polymethyl methacrylate is shown at the wavenumber, which is clearly distinguishable from the background spectrum. The signal-to-noise ratio is about 7.0 after BM4D denoising. In contrast, on the same sample and at the same average laser power, stimulated Raman scattering measurements did not show image contrast of 100-nanometer microspheres. Overall, compared with stimulated Raman scattering, stimulated Raman photothermal analysis shows improved sensitivity in both liquid samples and nanoparticle samples. Importantly, the introduction of a third detection beam with a shorter wavelength helps to improve spatial resolution. Figure 3D As shown in the figure, the intensity distribution across a pair of 100-nm polymethyl methacrylate microspheres was plotted, and the full width at half maximum (FWHM) of the Gaussian fit was found to be approximately 218 nm. Deconvolution, factoring in the microsphere size, yielded a FWHM of approximately 194 nm, which is below the theoretical resolution limit of stimulated Raman scattering under the same conditions (approximately 217 nm, the FWHM of the Airy disk).

[0071] See now Figures 4A to 4L, showing multiple images and graphs related to stimulated Raman photothermal imaging of biological samples in an aqueous environment according to the system and method described in the present disclosure. To explore the potential of stimulated Raman photothermal imaging in biological imaging, label-free stimulated Raman photothermal imaging of living SJSA-1 osteosarcoma cancer cells was first performed. Figure 4A As shown in image 400, lipid droplets, endoplasmic reticulum (ER) and nucleolus are at 2930 cm -1 The nuclear membrane shows a clear contrast, indicating that the photothermal intensity is locally enhanced in the aqueous environment. At an imaging speed of approximately 2.8 seconds per frame (s / frame), the dynamic process of lipid droplets is captured, and its entire trajectory is shown in Figure 2. Figure 4B As shown in image 406.

[0072] Next, hyperspectral stimulated Raman photothermal imaging (hyperspectral SRP imaging) was performed on SJSA-1 cells in the C–H stretching vibration region. Figure 4C As shown in image 412, subcellular structures such as lipid droplets and nucleoli show good image contrast. Phase analysis is then applied to segment the subcellular structures, where the five main components ( Figure 4D The image 418 in FIG. 4 is identified, along with the corresponding spectrogram.

[0073] To explore the applicability of stimulated Raman photothermal imaging in the "silent window" region, the cellular uptake of deuterated palmitic acid (PA-d31) was studied. Hyperspectral stimulated Raman photothermal images of SJSA-1 cells incubated with deuterated palmitic acid were generated. By phase analysis, the deuterated palmitic acid-enriched regions (cell membrane and endoplasmic reticulum) could be clearly separated from other cellular components ( Figure 4E 424 in the image). The spectral distribution obtained from the phase analysis ( Figure 4F 430) at 2100 cm -1 The peak value is at , which corresponds to C–D stretching vibration. In the control sample not treated with deuterated palmitic acid, only the signal of cellular components was observed ( Figure 4G Image 436 and Figure 4H442 in Figure 442). It is noteworthy that the background signal is unrelated to the Raman shift. This background signal is likely derived from overtone absorption of the C–H vibrational transition, which also deposits energy and produces photothermal contrast. The cell background signal in the C–D stimulated Raman photothermal image is stronger than that in the C–H stimulated Raman photothermal image ( Figure 4D ), which may be attributed to the 855 nm pump laser used for C–D excitation being close to the 920 nm overtone absorption band of C–H stretching vibration.

[0074] Using mouse brain tissue sections as samples, the performance of stimulated Raman photothermal imaging on tissue samples was evaluated, e.g. Figures 4I to 4K As shown in images 448, 454, and 460. Three fields of view (FOV) were selected as representatives of typical brain tissue structures. The detailed structure of the myelin sheath can be clearly resolved, showing typical lipid-rich Raman spectral characteristics. Myelin sheaths with different orientations show completely different signal intensities, indicating that the stimulated Raman photothermal signal intensity is highly sensitive to the laser polarization direction. In image 454, closely packed cytoplasmic organelles are observed. Stimulated Raman photothermal spectroscopy ( Figure 4L The diagram in 466) shows that myelin (468) is at 2845 cm -1 There is a lipid peak at 2930 cm -1 A protein peak is present at 470, while the cytoplasm signal (470) is dominated by proteins. The stimulated Raman photothermal signal from the medium is weak and unrelated to the Raman shift. Overall, the experimental data show the potential of stimulated Raman photothermal imaging for chemical analysis of tissue samples with good spectral fidelity.

[0075] Reference Figures 5A to 5F , showing stimulated Raman photothermal imaging images and spectra of biological samples immersed in glycerol. Compared with water, glycerol, with its high thermo-optical coefficient and low heat capacity, offers the possibility of further improving the sensitivity of stimulated Raman photothermal microscopy. High stimulated Raman photothermal image contrast ( Figure 3C ), stimulated Raman photothermal imaging of virus particles was first evaluated. Figure 5A As shown in image 510 in FIG, a single varicella-zoster viral particle (about 180 nm in diameter) can be clearly distinguished from the background with a signal-to-noise ratio of about 20. Stimulated Raman photothermal spectroscopy of a single virus ( Figure 5B The line 520 in the graph is at 2950 cm -1 A peak appears at , indicating that the nucleic acid at the viral core contributes significantly to the signal.

[0076] Deuterated glycerol (glycerol-d8) was used as a mounting medium to improve the quality of stimulated Raman photothermal imaging of mammalian cells. Pancreatic cancer cell MIA PaCa-2 was selected as the test object ( Figure 5C (As shown in image 530 in

[15] ). Deuterated glycerol was used instead of phosphate-buffered saline (PBS) to soak the cells in order to enhance the contrast of stimulated Raman photothermal images. Stimulated Raman photothermal imaging in the high-wavenumber C–H vibrational region showed significant contrast between membranes and intracellular lipids. Phase analysis was then applied to segment the cellular structure, with up to six different components (e.g., Figure 5D 540 ). Notably, the nuclear membrane 542 is clearly distinguished from the cytoplasm 544 and nuclear matrix 546, highlighting the potential of stimulated Raman photothermal imaging for studying fine membrane structures. This high contrast is likely due to the membrane's high thermo-optical coefficient and low heat capacity.

[0077] After adding glycerol, the high sensitivity of stimulated Raman photothermal analysis also provides a way to obtain weak Raman bands in the fingerprint region. Figure 5E OVCAR-5 cancer tissue with a thickness of 10 μm is shown at 1650 cm -1 Stimulated Raman photothermal image 550 at , which corresponds to the Amide I band in proteins and the C=C vibration in lipids. By stacking hyperspectral images, high-quality spectra are extracted. Figure 5F In the graph 560 , lipids (region of interest 1 (ROI 1), graph line 562 ) and proteins (region of interest 2 (ROI 1), graph line 564 ) can be clearly distinguished.

[0078] Finally, a direct comparison of stimulated Raman photothermal (ST) and stimulated Raman scattering (SRS) was performed on the same field of view of a single SKOV3 cell in glycerol medium, using constant average laser power and dwell time. In the resulting images, representative cellular structures (lipid droplets, nuclei, and nucleoli) were clearly resolved. A direct comparison of the distribution maps showed a signal-to-noise ratio of 53 for STS, compared to 17 for STS. Furthermore, the STS image exhibited clearer contrast within the nuclear structure.

[0079] Therefore, as discussed in this article, this study confirmed the existence of the stimulated Raman photothermal effect through numerical simulations and experiments. Based on this stimulated Raman photothermal effect, a stimulated Raman photothermal microscope was constructed and demonstrated to have superior detection sensitivity and resolution compared to traditional stimulated Raman scattering microscopy. Furthermore, stimulated Raman photothermal imaging of various biological samples in aqueous and glycerol environments was demonstrated. Below, stimulated Raman photothermal imaging is compared with stimulated Raman scattering in terms of detection mechanism, spatial resolution, laser noise, and solvent effects.

[0080] Stimulated Raman scattering (SRS) microscopy typically measures the gain in a Stokes beam or the loss in a pump beam. Therefore, a high numerical aperture (NA) objective is required to maximize signal collection efficiency and minimize cross-phase modulation. In contrast, SRPS microscopy measures light scattering caused by thermal expansion of particles or refraction due to thermal lensing. Therefore, SRPS is better suited to light collection using low NA objectives or air condensers. Results show that using an air condenser with a 0.5 NA yields sharp SRPS images, while SRPS images are relatively noisier. Furthermore, SRPS maintains good spectral fidelity, while SRPS spectra are distorted by enhanced cross-phase modulation background. Quantitatively, the SRPS images demonstrate an approximately 21.2-fold improvement in signal-to-noise ratio and a 7.8-fold improvement in signal-to-background ratio (SBR) compared to SRPS images. The relaxation of the requirements for high NA oil immersion lens brings convenience to the application of stimulated Raman photothermal technology.

[0081] Compared to stimulated Raman scattering (SRS), stimulated Raman photothermal (ST) also has a slight advantage in spatial resolution. By introducing a probe laser, the ideal effective point spread function (PSF) is the product of the PSFs of all three lasers (i.e., the pump laser, the Stokes laser, and the probe laser). Furthermore, a much shorter wavelength of probe light can be selected, resulting in a much sharper STS effective PSF. In this disclosure, based on a 1.49 numerical aperture objective lens, the ideal resolution reaches approximately 167 nanometers when using a 765-nanometer laser as the probe light, and approximately 137 nanometers when measuring with a 522-nanometer laser. It is worth noting that achieving this ideal resolution improvement requires high frequencies, as heat diffusion within the modulation cycle increases the size of the thermal lens, thus affecting resolution. At very low modulation frequencies, the resolution drops to that of stimulated Raman scattering (SRS). At the current modulation frequency of 125 kHz, the spatial resolution has been significantly improved, from approximately 217 nanometers for stimulated Raman scattering to approximately 194 nanometers for STS.

[0082] The different measurement methods for stimulated Raman photothermal (SRT) and stimulated Raman scattering (SRS) also result in different noise sources in the measurements. In both measurements, the primary noise sources are the relative intensity noise and shot noise of the measured laser beams—the probe laser for SRT and the pump / Stokes laser for SRS. Due to these different noise sources, SRT offers two advantages. First, SRT is less sensitive to the laser noise of the ultrafast lasers used to stimulate SRS. Therefore, SRT can be achieved using noisy ultrafast lasers. Second, the power of both the pump and Stokes lasers can be increased without affecting the SRT measurement noise. Therefore, SRT potentially enables the application of noisy, high-power lasers in vibration imaging.

[0083] Compared to stimulated Raman scattering, a notable feature of stimulated Raman photothermal (SRT) is its dependence on the properties of the sample environment. Therefore, lipid-rich regions in cell images, such as lipid droplets and lipid bilayers in membranes, show enhanced contrast compared to stimulated Raman scattering images. This property also makes it possible to enhance the intensity of the SRT signal through carefully designed media, such as critical xenon, which has been demonstrated to provide approximately 400-fold enhancement in visible light photothermal microscopy. However, it should be noted that the sample-dependent nature of the SRT signal may pose a challenge to quantitative analysis in highly heterogeneous environments.

[0084] There is still room for improvement in stimulated Raman photothermal imaging performance. In one example, imaging speeds of 8 microseconds per pixel, or approximately 3 frames per second (FPS) for a 200 × 200 pixel image, were achieved. This imaging speed is primarily limited by the low modulation frequency of 125 kHz. By measuring the stimulated Raman photothermal effect induced by a single stimulated Raman scattering excitation pulse pair at a repetition rate of 1 MHz, significantly higher imaging speeds could be achieved. Under these conditions, the imaging speed is limited only by the sample cooling rate, which is typically on the order of 1 microsecond per pixel (µs / pixel). This would be sufficient to achieve near-video-rate stimulated Raman photothermal imaging. In terms of detection sensitivity, more elegant photothermal detection schemes, such as fluorescence detection or nanomechanical photothermal sensing, could be introduced to enhance signal strength. In terms of spatial resolution, the resolution can be further improved to sub-100-nm levels by replacing the detection laser with a shorter wavelength and combining it with imaging scanning microscopy technique.

[0085] Reference Figure 9 , another embodiment of a stimulated Raman photothermal microscope 900 according to the technical solution of the present disclosure is shown. The stimulated Raman photothermal microscope 900 can be similar to the microscope 200, unless otherwise shown or described. Specifically, the microscope 900 uses a probe beam 902 from a fiber laser 904 as a substitute for the probe beam 208 of the laser 201c. In addition, the microscope 900 uses a long working distance air condenser 912 for collecting the probe beam, which is described in more detail below.

[0086] Generally speaking, a modulated Stokes and pump beam 906 (from a Stokes and pump laser 908) excites a stimulated Raman photothermal signal from a sample 910, which is detected using a probe beam 902 emitted by a fiber probe laser 904. Probe beam 902 is used to sense the thermal effects induced in the sample 910 by the stimulated Raman gain and loss processes (from the combined beam 906). Therefore, the signal-to-noise ratio is much less dependent on the noise in the fiber laser 904. Therefore, there is no need to balance the detection system. Furthermore, the stimulated Raman photothermal signal is inherently phase-modulated and can therefore be collected by an air condenser 912.

[0087] Microscope 900 can employ a 765-nanometer wave probe laser (e.g., TLB6712-D, SpectralPhysics). Its emitted probe beam 902 is collinear with a pump and Stokes beam 906 (e.g., Picus Duo, Refined Laser Systems) via a polarizing beam splitter 920. Combined beam 922 can be scanned by a scanning unit 923 and then focused onto sample 910 via a high numerical aperture (NA) water lens 924 (e.g., UPlanApo 60XW, NA 1.2, Olympus). In forward detection, the output beam 926 is collected by an air condenser 930 (e.g., NA 0.55, Nikon), passes through a 770 nm bandpass filter 932 (e.g., FB770-10-Ø1", Thorlabs) and lens 934, and is detected by a silicon photodiode 936 to generate stimulated Raman thermal image contrast. Backward detection utilizes a dichroic mirror 940, a pinhole 942, and a silicon photomultiplier 946 for confocal fluorescence schemes and multimodal imaging. Many stimulated Raman scattering and stimulated Raman thermal setups utilize high numerical aperture objectives and oil immersion condensers on both sides of the sample. However, microscope 900 utilizes a long working distance air condenser 912 for forward detection, facilitating live cell imaging and large tissue area screening, and is compatible with most fluorescence setups.

[0088] Unlike stimulated Raman scattering imaging, which detects weak modulations in localized oscillators, stimulated Raman photothermal (SRT) uses a third beam (probe beam 902) to detect SRT heat deposition, making it inherently immune to fiber laser noise. Therefore, fiber laser-based SRT eliminates the need for balanced detection techniques. Furthermore, through sensitive thermal lens detection, SRT achieves higher sensitivity in media with high thermo-optical coefficients.

[0089] To demonstrate this, the performance of stimulated Raman scattering (SRS) and stimulated Raman photothermal (SRT) was compared in the detection of the C–H symmetric stretching vibration of dimethyl sulfoxide (DMSO). SRS was collected using a high-NA condenser (e.g., Aplanat Achromat 1.4, NA 1.4, Olympus), while SRS was collected using a low-NA air condenser. Without balancing, SRS provided a signal-to-noise ratio (SNR) of only 87.6, affected by the high laser noise in the detected pump beam. Using automated balancing, the SNR improved to 742. At this excitation power, SRS achieved a SNR of up to 3300, enabling highly sensitive detection of biomolecules.

[0090] In stimulated Raman scattering (SRS) microscopy, precise alignment and focusing of high-NA objectives and condensers are crucial. This precision setup is necessary to minimize light collection losses and prevent the introduction of non-vibrational background, such as cross-phase modulation (XPM), which can significantly distort spectral information. In contrast, the requirements for stimulated Raman photothermal microscopy, which relies on thermal lens detection, are different. SRS requires only a low-NA light collector, which simplifies the setup process and allows the use of an original long-working-distance air condenser. This simpler setup does not compromise the quality of SRS imaging, achieving high-quality images and maintaining spectral fidelity. In contrast, stimulated Raman scattering is often contaminated by parasitic cross-phase modulation background, resulting in loss of chemical information.

[0091] Fiber laser-based stimulated Raman photothermal technology, with its sensitivity and full-spectrum detection capabilities, offers new possibilities for biological applications. This new technology was applied to two biological samples: bladder cancer T24 cells and breast cancer tissue. Both samples were immersed in deuterated glycerol to enhance the thermal lensing effect. Imaging results demonstrated that fiber laser-based stimulated Raman photothermal technology can provide detailed subcellular distribution maps containing vibrational information, which can be used for chemical composition analysis and quantitative results. This method is also useful for screening large tissue areas, as demonstrated by mapping the distribution of biomolecules (such as lipids and proteins) in cancer tissue and by its broadband detection capabilities in fingerprint regions.

[0092] In summary, fiber laser-based stimulated Raman photothermal microscopy has demonstrated significant progress in the field of biological microscopy, providing higher sensitivity, full-spectrum detection, and user-friendly operation, and is expected to bring about changes in the field of biological imaging and analysis.

[0093] All documents cited herein are incorporated by reference as if fully set forth herein. The orientation and arrangement of all components shown herein are exemplary only. Furthermore, it will be understood by those skilled in the art that multiple functional elements may be implemented by fewer elements or a single element in alternative embodiments. Similarly, in certain embodiments, any functional element may perform fewer or different operations than those described in the embodiments. Furthermore, independent functional elements shown for illustrative purposes may also be integrated into other functional elements in specific implementations.

[0094] Although the present invention has been described based on preferred embodiments, those skilled in the art will readily appreciate that various changes and / or modifications may be made to the present invention without departing from the spirit and scope of the present invention. For example, each claim may be subject to multiple dependencies on any or all other claims, even if not drafted in this manner in the original application.

Claims

1. A stimulated Raman photothermal microscope for sample imaging, comprising: a first light source, wherein the first light source omits an intensity-modulated pump beam; a second light source omitting an intensity-modulated Stokes beam, wherein the Stokes beam is combined with the pump beam to form a composite beam, the composite beam being directed to the sample to induce a heating effect caused by a stimulated Raman process; a third light source that emits a probe beam, wherein the probe beam is directed toward the sample; as well as A light detector is configured to detect the modulation of the probe light beam after the probe light beam is modulated by the sample, so as to measure a stimulated Raman photothermal signal.

2. The microscope of claim 1, wherein the probe beam is directed to the sample co-linearly with the composite beam.

3. The microscope according to claim 2 further includes at least one lens located in the optical path of the composite light beam and the detection light beam, and the at least one lens adjusts the collimation of the detection light beam so that the focus of the detection light beam deviates axially from the focus of the composite light beam.

4. The microscope of claim 1 , wherein the duty cycle of the probe beam is between 5% and 10%.

5. The microscope of claim 1, wherein the microscope further comprises a glycerol-based medium for mounting the sample. The microscope of claim 1 , wherein the probe beam has a wavelength different from that of the pump beam and the Stokes beam.

7. The microscope of claim 6, further comprising at least one scanning mirror configured to scan the sample with the composite beam and the probe beam.

8. The microscope of claim 1, wherein the modulation frequency of the probe beam is at least 125 kHz.

9. The microscope according to claim 1 further comprises a spectral filter, wherein the light detector detects the probe light beam after the probe light beam is modulated by the sample and filtered by the spectral filter.

10. The microscope of claim 1, wherein the measured stimulated Raman photothermal signal is based on a local refractive index modulation of the sample and is determined by the detected probe beam.

11. The microscope of claim 1 , wherein the third light source is a fiber laser.

12. The microscope according to claim 11, further comprising an air condenser, wherein the air condenser is configured to collect the probe light beam after the probe light beam is modulated by the sample and before the probe light beam is detected by the light detector.

13. A stimulated Raman photothermal microscope for sample imaging, comprising: a first light source, wherein the first light source omits an intensity-modulated pump beam; a second light source omitting an intensity-modulated Stokes beam, wherein the Stokes beam is combined with a pump beam to form a composite beam, the composite beam being directed to the sample to induce a heating effect caused by the stimulated Raman process; a fiber laser that emits a probe beam directed toward the sample; as well as A photodiode is configured to detect the modulation of the probe light beam after the probe light beam is modulated by the sample, so as to measure a stimulated Raman photothermal signal.

14. The microscope according to claim 13, wherein: The photodiode is part of a forward detection system; The microscope further comprises a rearward detection system, wherein the rearward detection system comprises a pinhole and a photomultiplier tube; The duty cycle of the probe beam is between 5% and 10%.

15. The microscope according to claim 13, further comprising an air condenser, wherein the air condenser is configured to collect the detection beam after the detection beam is modulated by the sample and before it is detected by the photodiode.

16. The microscope of claim 13, wherein: The probe beam is directed to the sample collinearly with the composite beam; and The microscope further includes at least one lens located in the optical path of the composite beam and the probe beam, the at least one lens adjusting the collimation of the probe beam so that the focus of the probe beam is axially offset from the focus of the composite beam.

17. The microscope of claim 13, wherein the microscope further comprises a glycerol-based medium for mounting the sample.

18. The microscope of claim 13, wherein: The probe beam has a wavelength different from that of the pump beam and the Stokes beam; and The microscope further comprises at least one scanning mirror configured to scan the sample using the composite beam and the probe beam.

19. The microscope of claim 13, further comprising a spectral filter, wherein the light detector detects the probe beam after the probe beam is modulated by the sample and filtered by the spectral filter.

20. The microscope of claim 13, wherein the measured stimulated Raman photothermal signal is based on a local refractive index modulation of the sample and is determined by the detected probe beam.

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