CN118010700A - Cross-modal spectroscopy and imaging method and device based on single beam detection - Google Patents

Abstract

The invention discloses a method and a device for cross-modal spectrum and imaging based on single-beam detection. The method and the device are suitable for the fields of optical imaging and molecular vibration spectrum imaging, and the device comprises a light source module, a microscope module, a detection module and a spectrum and imaging acquisition module. The technology utilizes the fact that two physical processes of stimulated radiation and photo-thermal relaxation have obvious time characteristic differences, and modulation is carried out in respective time windows, so that the highest modulation efficiency and the detection effect of the minimum crosstalk are achieved. The infrared and Raman signals are carried in the light intensity of a single light beam by a pumping detection technology, and the infrared and Raman signals are separated by a frequency domain filtering technology. The method solves the technical problem that the infrared spectrum and the Raman spectrum are difficult to integrate on the same spectrum acquisition and imaging system due to the difference of optical properties and different detection schemes. The simultaneous spectrum acquisition and imaging of infrared and Raman spectrum modes are realized.

Description

Cross-modal spectroscopy and imaging method and device based on single beam detection

Technical Field

The present invention belongs to the field of optical imaging and molecular vibration spectrum imaging, and in particular relates to a method and device for cross-modal spectrum and imaging based on single-beam detection.

Background technique

The goal of cross-modal imaging is to provide unique and complementary insights into various targets by synergistically combining different microscopic imaging modes, providing a holistic view of molecular dynamics for comprehensive chemical, physical, and biological analyses. Compared with single imaging methods, cross-modal imaging can provide additional molecular spatial information. For example, the introduction of fluorescence imaging modes to complement the lack of specificity of digital holography and interferometric scattering imaging has demonstrated the power of cross-modal imaging in studying organelle interactions and tracking nanoscale dynamic events. Faced with the challenges of labeling small molecules and metabolites, label-free imaging methods have been developed to complement the molecular species limitation in fluorescence imaging modalities by capturing inherent molecular structural information. Notably, nonlinear optical processes such as second harmonic generation (SHG), transient absorption (TA), stimulated Raman scattering (SRS), and third-order sum frequency generation (TSFG) are compatible with each other and provide new insights into cellular metabolism and pathological mechanisms. In addition, by combining multiple imaging modalities such as scattering, photoacoustic, phase, and fluorescence, pump-probe techniques based on the photothermal effect induced by light absorption have demonstrated significant synergistic effects, greatly improving the resolution, sensitivity, and throughput of label-free vibrational imaging.

In order to non-invasively detect the intrinsic fingerprint information of molecular vibration, infrared and Raman spectroscopy have been widely used in various fields, especially infrared spectroscopy, whose absorption cross section (about ^{10-18 cm2} ) is much higher than that of conventional Raman spectroscopy ( ^{10-28 cm2} ). However, it is difficult to determine the complete molecular structure information from a single spectral mode, because the vibration mode with Raman activity is not necessarily infrared active, and vice versa. The infrared spectrum is very sensitive to asymmetric vibrations, while the Raman spectrum is sensitive to symmetric vibrations, which leads to incomplete spectral information. Therefore, it is necessary to combine infrared spectroscopy and Raman spectroscopy for simultaneous detection to provide complementary and comprehensive vibrational spectroscopy information, so as to have a deeper understanding of complex processes. For example, by integrating infrared spectroscopy and Raman spectroscopy measurements, it has been demonstrated in the field of chemistry that it can identify new _NOx adsorption states and visualize information related to the location of _NOx storage efficiency in heterogeneous catalytic processes. Recent studies on CO reduction reactions have used in situ enhanced infrared and Raman spectroscopy techniques to reveal the formation mechanism of CO subpopulations during adsorption. Early attempts to sequentially image biological samples using infrared and Raman spectroscopy have shown their potential to provide a full spectrum of cellular composition and enhance biomarker identification.

However, it is not feasible to directly combine the infrared and Raman modalities because they have different physical properties (absorption vs. scattering) and significant differences between the spectral windows (mid-infrared vs. visible light). Schemes for combined infrared and Raman detection have been proposed. For example, IRaman combines mid-infrared photothermal imaging with single-point Raman spectroscopy. To overcome the conflict of infrared and Raman modal selection rules, nonlinear optical processes have been introduced, including two-photon excited Raman spectroscopy, namely hyper-Raman spectroscopy. In addition, there are single-point spectral measurements of complementary vibrational spectra through Fourier transform coherent anti-Stokes Raman scattering (Raman sensitive) and intra-pulse difference frequency generation (infrared sensitive) processes. The three-photon process combining coherent anti-Stokes Raman scattering and third-order sum frequency generation improves the resolution of infrared and Raman imaging. However, it remains a huge challenge to develop a simultaneous infrared and Raman imaging platform with high-resolution and quantitative chemical imaging capabilities.

In summary, there is still a lack of a solution that can simultaneously perform spectroscopy and imaging of infrared and Raman modes, and an effective solution has not yet been found. This problem requires further research and development to meet the needs of the optical imaging field for simultaneous spectroscopy and imaging of infrared and Raman modes.

Summary of the invention

Based on the technical bottlenecks mentioned above, the present invention provides a cross-modal spectroscopy and imaging method based on single-beam detection, which can achieve label-free, high-sensitivity, and high-spectral range simultaneous infrared and Raman spectroscopy and imaging acquisition.

The present invention first provides a method for cross-modal spectroscopy and imaging based on single-beam detection, comprising the following steps:

1) regulating a beam of stimulated emission light and a beam of wavelength-tunable pump light so that the two beams overlap in time and space, and then adjusting a beam of wavelength-tunable mid-infrared light so that the optical path of the mid-infrared light is collinear with the stimulated emission light and the pump light, and further focusing the three beams so that the focal points of the three beams overlap in space;

2) Setting the target sample to be detected at the focus of the three beams;

3) adjusting the wavelength of the pump light so that the energy difference between the pump light and the stimulated radiation light matches the Raman vibration energy level of the target molecule to achieve a stimulated Raman scattering effect, and adjusting the wavelength of the mid-infrared light so that the mid-infrared light matches the infrared vibration energy level of the target molecule to achieve a localized photothermal relaxation effect around the target molecule, so that the target molecules within the focus of the three beams simultaneously produce a stimulated Raman scattering effect and a mid-infrared photothermal relaxation effect produced by mid-infrared absorption; thereby causing the stimulated Raman scattering signal produced by the stimulated Raman scattering effect and the mid-infrared photothermal signal produced by the mid-infrared photothermal relaxation effect to be carried in the pump light intensity;

4) Using the difference in the time characteristics of the stimulated Raman scattering effect and the photothermal relaxation effect, the intensity of the stimulated radiation light and the mid-infrared light is modulated in the time domain, so that the signals of the stimulated Raman scattering effect and the photothermal relaxation effect are in different frequency ranges;

5) After the stimulated Raman scattering signal and the mid-infrared photothermal signal generated in different frequency intervals are carried by the pump light, the pump light is input into the photodetector as the detection light and converted into a photovoltage signal, and the stimulated Raman scattering signal and the mid-infrared photothermal signal are separated from the photovoltage signal by using the frequency domain filtering technology;

6) Scan the spatial position of the target sample and simultaneously collect stimulated Raman scattering signals and mid-infrared photothermal signals to realize the collection of cross-modal imaging data. The collected cross-modal imaging data is processed by spectral and imaging processing equipment to generate an imaging image;

7) Scanning the wavelength of the pump light and the wavelength of the mid-infrared light, collecting spectra of the molecules within the common focus of the stimulated radiation light, the pump light, and the mid-infrared light to obtain spectra of each mode.

As a preferred embodiment of the present invention, in step 1), the stimulated radiation light and the pump light are ultrafast lasers with a pulse width of femtosecond to picosecond order and a wavelength range from visible light to near-infrared light; the wavelength of the pump light is shorter than that of the stimulated radiation light, and the photon energy difference between the stimulated radiation light and the pump light covers the molecular vibration spectrum range.

As a preferred embodiment of the present invention, the stimulated radiation light and the pump light in step 1) need to be adjusted to overlap in the time domain; the pulsed mid-infrared light does not overlap with the stimulated radiation light and the pump light in the time domain to avoid nonlinear effects with the stimulated radiation light and the pump light.

The present invention also provides a cross-modal spectroscopy and imaging device for implementing the above cross-modal spectroscopy and imaging method, comprising:

A light source module, comprising a first light source for emitting stimulated radiation light and pump light, a second light source for emitting mid-infrared light, an electro-optic modulator for performing time domain modulation on the stimulated radiation light, and an optical path module for performing spatial domain coincidence adjustment on the stimulated radiation light and the pump light and collinear adjustment on the mid-infrared light;

A microscope module, including a microscope frame, a lens module for adjusting light, an infrared focusing mirror for focusing mid-infrared light and collecting pump light, and a sample scanning stage for carrying samples;

A detection module, including a collection optical path for collecting signals, a filter set for filtering out pump light, a photodetector for converting the intensity of the pump light into a photovoltage signal, and a preamplifier;

The spectrum and imaging acquisition module comprises a data acquisition card, a filter device and a spectrum and imaging processing device; the data acquisition card and the filter device are used to acquire cross-modal imaging data and spectra; the spectrum and imaging processing device is used to control the data acquisition card, the sample stage, the first light source, the second light source and the filter device.

As a preferred embodiment of the present invention, the lens module includes a first wide-beam lens, a second wide-beam lens, a third wide-beam lens, a fourth wide-beam lens, a first reflector, a second reflector, a third reflector 8, a dichroic mirror, a fourth reflector, a 50-50 beam splitter, a fifth reflector, a beam splitter, a sixth reflector, a seventh reflector, a refracting objective lens and an infrared focusing lens.

Compared with the prior art, the present invention has the following beneficial effects:

(1) By utilizing the time scale differences of different physical processes to perform time domain modulation, the technical difficulty of integrating infrared spectroscopy and Raman spectroscopy into the same spectral acquisition and imaging system due to the large differences in optical properties and different detection schemes is fundamentally solved, and the difficult simultaneous imaging of infrared and Raman complementary spectral modes is realized.

(ii) The present invention not only gets rid of the limitations of low resolution of infrared imaging, water absorption, weak signal of Raman spectrum, etc., but also allows photothermal imaging and stimulated Raman scattering imaging to be detected on the same detection light, thereby greatly improving the resolution and sensitivity of infrared imaging and the sensitivity and speed of Raman imaging.

(III) The present invention uses a single detection beam and a single detector to simplify the system complexity, avoid the crosstalk problem of traditional multi-modal imaging, and achieve cross-talk-free imaging. It also improves the imaging flux and achieves cross-spectral imaging capabilities.

(iv) The present invention realizes the simultaneous imaging of different physical processes from picoseconds to microseconds, and has the characteristics of mutual compatibility and universality. In principle, this scheme is applicable to any multimodal imaging method based on time domain modulation.

(V) The present invention is generally applicable to all molecules and is not limited to mid-infrared absorption. Benefiting from the photothermal detection scheme, the present invention is applicable to any imaging modality based on absorption detection; and provides more spectral windows for various imaging modes of picosecond ultrafast laser excitation, and is compatible with a variety of ultrafast optical modalities, such as two-photon fluorescence, transient absorption, coherent anti-Stokes Raman imaging, second harmonic imaging, third harmonic imaging, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

FIG1 is an energy level schematic diagram of cross-modal spectroscopy and imaging for single-beam detection;

FIG2 is a schematic diagram of a portion of an experimental optical path according to an embodiment of the present invention;

FIG3 is a signal transmission and system control diagram of an embodiment of the present invention;

FIG4 is a waveform diagram of an incident laser pulse, including pump light, stimulated radiation light and mid-infrared light pulse in the time domain according to an embodiment of the present invention;

FIG5 is a diagram showing the intensity of the detection light in the time domain according to an embodiment of the present invention;

FIG6 is a diagram showing the intensity of the detection light in the frequency domain according to an embodiment of the present invention;

FIG7 is a spectrum diagram detected on an oil film according to an embodiment of the present invention;

FIG8 is a graph showing infrared and Raman spectra of 500 nm PMMA microspheres measured simultaneously according to an embodiment of the present invention;

FIG9 is an infrared and Raman image of 500 nm PMMA plastic microspheres collected simultaneously according to an embodiment of the present invention;

FIG10 is a cross-section of infrared and Raman signals of a single microsphere indicated by an arrow in FIG9 and a Gaussian fitting result diagram;

FIG11 is a three-dimensional infrared image of a single 500nm PMMA plastic microsphere collected in an embodiment of the present invention;

FIG12 is a three-dimensional Raman image of a single 500 nm PMMA plastic microsphere collected simultaneously with FIG11 according to an embodiment of the present invention;

FIG13 is a 3D reconstruction of the infrared and Raman images of a single 500 nm PMMA plastic microsphere of FIG11 and FIG12 ;

FIG14 is a longitudinal section of a single 500 nm PMMA plastic microsphere 3D imaging of FIG11 and FIG12 and a Gaussian fitting result diagram;

FIG15 is an infrared transmission and spontaneous Raman spectra of palmitic acid (PA), 1,4-diphenylbutadiyne (DiPhDY) and triphenylphosphine (TPP) used in the examples of the present invention;

FIG16 shows the infrared and Raman activities of three chemical substances used in the embodiments of the present invention at different wave numbers;

FIG17 is an infrared and Raman image of a mixture of three chemical substances mixed in equal proportions collected simultaneously in an embodiment of the present invention;

FIG18 is a pure substance distribution diagram separated from the data of FIG17 and an overlapped diagram of the distribution diagrams of three substances;

FIG19 is a schematic diagram showing the Pearson correlation coefficient between the imaging images shown in FIG17 ;

FIG20 is an image of cells in the control group and the experimental group collected simultaneously under infrared 1750 cm ^-1 and Raman 2850 cm ^-1 according to an embodiment of the present invention;

FIG21 is a ratio imaging diagram of cells in the control group and the experimental group at infrared 1750 cm ^-1 and Raman 2850 cm ^-1 ;

FIG22 is a distribution histogram of the number of lipid droplets in cells of the control group and the experimental group as a function of relative intensity;

FIG23 is an image of cells in the control group and the experimental group simultaneously collected at infrared 1080 cm ^-1 and Raman 2930 cm ^-1 in an embodiment of the present invention;

FIG24 is a ratio imaging diagram of cells in the control group and the experimental group at infrared 1080 cm ^-1 and Raman 2850 cm ^-1 and their local magnified diagrams;

FIG25 is a distribution diagram of the area and relative intensity of polysaccharide protein particles in cells of the control group and the experimental group;

FIG26 is a schematic diagram showing the Pearson correlation coefficient between the imaging images of the control group and the experimental group cells shown in FIG20 and FIG21 ;

FIG27 shows back infrared and forward Raman images of the striatum in the mouse brain collected simultaneously according to an embodiment of the present invention;

FIG28 is an in-situ infrared and Raman spectra of three locations of interest indicated by arrows in FIG27 ;

FIG29 shows an infrared image of Caenorhabditis elegans collected by an embodiment of the present invention;

FIG30 shows a Raman image of Caenorhabditis elegans collected according to an embodiment of the present invention.

Detailed ways

In general, the embodiments described herein relate to the use of frequency multiplexing technology to achieve single beam detection of cross-modal spectral information to obtain multi-spectral images. As required, the implementation method of the present invention is disclosed herein. However, it should be noted that the implementation method involved in this article is only exemplary, so the present invention can be implemented in many different and alternative forms.

Moreover, in order to highlight the novel aspects of the present invention and to take into account the simplicity of the drawings, some of the schematic diagrams in the drawings shown in this article are not necessarily in actual proportion, and some features may be exaggerated or minimized to show the details of specific things. At the same time, in the drawings of this article, especially the device diagrams, related elements may have been omitted to prevent blurring the many innovative aspects of the new device. Therefore, the methods, structures, and functional details disclosed in this article should not be understood as limitations, but only as the basis for the claims and as a representative basis for teaching researchers in the field to use the present invention in a variety of ways. For the purpose of guidance rather than limitation, this article discloses a method for obtaining multi-spectral images by using frequency multiplexing technology to detect cross-modal spectral information with a single beam.

The detection principle of the present invention can be explained through an energy level diagram. Molecules with specific structures have Raman vibration energy levels and infrared vibration energy levels, as shown in FIG1 . A beam of pump light and a beam of stimulated radiation light are introduced to irradiate a specific molecule, and the molecule will be excited to a virtual energy level by the pump light. When the energy difference between the pump light and the stimulated radiation light matches the energy of Raman vibration, the introduced stimulated radiation light will produce stimulated radiation on the Raman photons, which will amplify the Raman signal by about 10 ⁸ times. The amplified Raman signal is called stimulated Raman signal: the pump light intensity will carry a stimulated Raman loss SRL signal, and the stimulated Raman gain SRG signal will be carried on the intensity of the stimulated radiation light. When a beam of mid-infrared light is used to irradiate the molecule, but the energy of the mid-infrared light matches the energy of the infrared vibration energy level, the molecule will directly absorb the infrared light energy. The absorbed energy will cause the surrounding temperature to rise after dissipation, thereby changing the local refractive index and causing deformation. At this time, a significant photothermal effect is generated, which will modulate the propagation of the light beam irradiated at the same spatial position through a photothermal lens, which is called a mid-infrared photothermal MIP signal. At this time, the infrared spectrum is linearly related to the MIP signal intensity.

Therefore, it is reasonable to propose a pump-probe scheme to detect these two processes simultaneously. When the three beams mentioned above are simultaneously hit on the sample, the intensity of the pump light and the stimulated radiation light will simultaneously generate stimulated Raman scattering signals and photothermal signals. This detection method can bring two completely different modes into the same dimension for detection.

The stimulated Raman process and photothermal process mentioned above, as shown in Figure 1, will occur when the pump light, stimulated radiation light and mid-infrared light are continuous light and pulsed light. The present invention takes advantage of the fact that the stimulated Raman process and the photothermal process have completely different characteristics in terms of time scale: the stimulated Raman scattering process occurs in the time scale range of femtoseconds to picoseconds, and the photothermal relaxation process occurs in the process of nanoseconds to microseconds. A pulsed light excitation detection scheme is adopted, and the stimulated Raman scattering process and the photothermal relaxation process are modulated in their respective time windows to achieve minimum signal crosstalk and maximum modulation efficiency, that is, to achieve cross-modal spectral imaging of single-beam detection.

In addition, both pump light and stimulated radiation light carry stimulated Raman scattering signals and mid-infrared photothermal signals, and can be used as detection light to achieve cross-modal spectral imaging of single-beam detection. Considering many factors, such as the detection quantum efficiency and spot diffraction limit size of conventional silicon-based detectors, the present invention selects pump light as the detection light to achieve better performance, including higher detection sensitivity and higher imaging resolution.

In a specific embodiment of the present invention, as shown in FIG2, a cross-modal spectroscopy and imaging device based on single beam detection disclosed by the present invention includes a first light source 1, a first wide beam lens 2, a second wide beam lens 3, a third wide beam lens 4, a fourth wide beam lens 5, a first reflector 6, a second reflector 7, a third reflector 8, an electro-optical modulator 9, a dichroic mirror 10, a fourth reflector 11, a 50-50 beam splitter 12, a second light source 13, a fifth reflector 14, a beam splitter 15, a sixth reflector 16, a seventh reflector 17, a refractive objective lens 18, an infrared focusing lens 19, a sample scanning stage 20, a first filter set 21, a first photodetector 22, a second filter set 23, and a second photodetector 24. The process of implementing cross-modal spectroscopy and imaging of single beam detection using the system shown in FIG2 and FIG3 is as follows:

1) The first light source 1 is an ultrafast laser light source, emitting two laser beams with a pulse width of 2 picoseconds and a basic pulse repetition frequency of 80 MHz. One of the beams is a wavelength-tunable pump light with a tunable range of 700-990 nanometers, which is adjusted to parallel light through the first wide-beam lens 2 and the second wide-beam lens 3, and then propagates through the dichroic mirror 10. The other beam is a stimulated radiation light with a wavelength fixed at 1031 nanometers, which is collimated and adjusted to parallel light by the third wide-beam lens 4 and the fourth wide-beam lens 5, and then guided to the electro-optic modulator 9 through the first reflector 6, the second reflector 7, and the third reflector 8 in sequence, modulated to a frequency of 20 MHz, and reflected by the dichroic mirror. The stimulated radiation light and the pump light are combined by adjusting the angles of the three reflectors.

2) The optical path of the stimulated radiation light is adjusted by moving the distance between the second reflector 7 and the third reflector 8 to adjust the time overlap of the pump light and the stimulated radiation light pulse focused on the sample; the moving direction is indicated by the arrow, and the stimulated Raman scattering effect reaches the highest signal value when the time delay between the stimulated radiation light and the pump light is 0 picoseconds. By adjusting the time delay between the stimulated radiation light and the pump light pulse, the detection of time-resolved coherent Raman scattering can be achieved.

3) The combined pump light and stimulated radiation light will be guided by the reflector 11, transmitted through the 50-50 beam splitter 12, and focused on the sample by the refractive objective lens 18; the wavelength-tunable mid-infrared light emitted by the second light source 13 has a tunable wavelength range of 5-10.5 microns, and the repetition frequency is adjusted to 100 kHz by electrically modulating the laser, and the pulse width is 500 nanoseconds, and then guided by the fifth reflector 14 and transmitted through the beam splitter 15;

4) The mid-infrared light is then guided to the infrared focusing mirror 19 through the sixth reflector 16 and the seventh reflector 17 and focused on the sample. By adjusting the angle between the two reflectors and the spatial position of the infrared focusing mirror, the optical axis of the mid-infrared light path is completely overlapped with the optical axis of the pump light and the stimulated radiation light, and the focal spot of the mid-infrared laser is spatially overlapped with the focal spot of the pump light and the stimulated radiation light;

5) The sample is mounted on the sample scanning stage 20. By tuning the wavelength of the pump light so that the energy difference between the pump light and the stimulated radiation light matches the corresponding Raman vibration energy level, the wavelength of the mid-infrared light is tuned to the infrared vibration energy level. The sample generates a corresponding stimulated Raman scattering signal and a mid-infrared photothermal signal, and the signal will be carried in the pump light and the stimulated radiation light;

6) The pump light carrying the stimulated Raman scattering signal and the mid-infrared photothermal signal is used as the detection light, which can be detected in the backward or forward direction. The forward direction is the incident direction of the pump light, and the backward direction is the direction opposite to the incident direction of the pump light. The process of backward detection is: after being collected by the refractive objective lens 18, it is reflected by the 50-50 beam splitter, and then filtered out by the first filter group 21 to be incident on the first photodetector 22; the process of forward detection is: forward collection is performed through the infrared focusing mirror 19, guided by the seventh reflector 17 and the sixth reflector 16 in turn, and separated from the optical path by reflection by the beam combiner 15, and then filtered out by the second filter group 23 to be incident on the second photodetector 24; when performing forward detection, the infrared focusing mirror used to collect the pump light needs to maintain high transmittance and collection efficiency for the pump light to reduce the loss of the pump light during detection.

7) After the photodetectors 22 and 24 convert the obtained optical signals into electrical signals, they are input into the preamplifier 28 for further voltage signal amplification, and then input into the filter device 26 for digital-to-analog conversion. The filter device receives the modulation frequency signal of the second light source 13 and the modulation frequency signal of the electro-optical modulator 9 as the center frequency band of filtering, and performs narrow-band filtering at 100 kHz and 20 MHz at the same time to separate the signal components, that is, the stimulated Raman scattering signal and the mid-infrared photothermal signal are detected at the same time;

8) Performing spatial scanning through the sample stage 20 and simultaneously collecting the output signal of the filter device to realize cross-modal infrared and Raman imaging data collection based on single-beam detection, and the collected data is pixel-segmented and averaged through the spectrum and imaging processing device 25 to form an imaging image; the imaging speed, field of view, pixel size and collection time are controlled by the spectrum and imaging processing device 25;

9) By scanning the wavelength of the mid-infrared light emitted by the second light source 13 and the wavelength of the pump light emitted by the first light source 1, spectrum collection is performed on the molecules within the common focus of the stimulated radiation light, the pump light, and the mid-infrared light, so as to realize cross-modal in-situ infrared and Raman spectroscopy data collection based on single-beam detection, and the cross-modal in-situ infrared and Raman spectroscopy collection band and speed are controlled by the spectrum and imaging processing device 25;

In order to more conveniently understand the detection scheme of the present invention, the pulse sequence diagram of the incident pump light, stimulated radiation light, and mid-infrared light shown in Figure 4 is combined, where f _P , f _S , and f _M represent the modulation frequencies of the pump light, stimulated radiation light, and mid-infrared light, respectively. In one embodiment of the present invention, f _P , f _S , and f _M are 80 MHz, 20 MHz, and 100 kHz, respectively. The propagation of the wavelength-tunable pump light with a pulse width of 2 picoseconds and a repetition frequency of 80 MHz produces photothermal modulation. The wavelength-tunable pump light intensity with a repetition frequency of 80 MHz encodes a group of stimulated Raman loss signals with a modulation frequency of 20 MHz at a high frequency and a group of photothermal signals with a modulation frequency of 100 kHz at a low frequency. The pump light intensity is converted into a photovoltage signal by a photodetector, and the photovoltage signal is input into the filter device after preamplification. In order to more conveniently understand the signal composition generated by this embodiment, in combination with FIG. 5 and FIG. 6, the characteristics of the waveform of the detection light in the time domain and the frequency domain are described. The present invention selects pump light as the detection light to achieve better performance. As shown in FIG. 5, the pump light waveform in the time domain carries the SRL signal and the MIP signal. As shown in FIG. 6, the frequency domain component composition of the detection light intensity in FIG. 5, the SRL signal carried is presented as a peak at a frequency of f _S , and the MIP signal carried is presented as a series of harmonic peaks with a fundamental frequency of f _M. In this embodiment, the infrared light modulation frequency and the stimulated radiation light modulation frequency are at a frequency spectrum distance of f _S -f _M , which is 19.9 MHz, and can avoid mixing of MIP signals and SRS signals. While eliminating signal crosstalk, high-frequency modulation can avoid low-frequency noise and significantly improve the signal-to-noise ratio. FIG. 7 shows the frequency domain composition of the signal actually measured on the olive oil sample. No frequency component is observed at f _S ±f _M , which indicates that there is no crosstalk between signals in the cross-modal spectrum and imaging method of single-beam detection.

In a specific embodiment of the present invention, a cross-modal spectroscopy and imaging method using a single beam detection has been experimentally implemented, and cross-modal spectral data and imaging data have been collected for 500-nanometer polymethyl methacrylate PMMA plastic microspheres. FIG8 shows the infrared and Raman spectral data. Using the collected spectral data, the C=O stretching vibration band with an infrared mode of 1730 cm ^-1 and the _CH3 asymmetric stretching vibration band with a Raman mode of 2957 cm ^-1 were selected to perform cross-modal imaging with a single beam detection, and the results are shown in FIG9. FIG10 shows a cross-sectional view of the imaging of a 500-nanometer microsphere by the present invention, and the resolution of infrared and Raman imaging can be calculated by the deconvolution method. The resolution of infrared imaging is 599 nanometers, and the resolution of Raman imaging is 490 nanometers, which is slightly better than that of infrared imaging, mainly because Raman imaging is based on a nonlinear stimulated Raman scattering scheme, and the resolution is jointly determined by the pump light and the stimulated radiation light, while the resolution of infrared imaging is mainly determined by the pump light.

In addition, a single microsphere was subjected to three-dimensional imaging of the transmembrane state by single-beam detection, as shown in Figures 11 and 12, which are images collected at infrared 1730 cm ^-1 and Raman 2957 cm ^-1 , respectively, and its three-dimensional reconstruction is shown in Figure 13. Figure 14 is an axial cross-sectional view in Figure 13. Through three-dimensional imaging, it can be seen that the imaging images of the two modes do not have a focal plane offset, which further ensures the reliability of the transmembrane state imaging system provided by the present invention and ensures the feasibility of quantitative analysis of cross-modal data.

In order to demonstrate the enhanced chemical specificity of the present invention, in a specific embodiment of the present invention, single beam detection transmembrane state imaging of three mixtures including palmitic acid PA, 1,4-diphenylbutadiyne DiPhDY and triphenylphosphine TPP was performed, including a similar structure and overlapping spectral band diagram as shown in Figure 15. Since the present invention has a broad and independent infrared and Raman spectral coverage, chemical bands with higher specificity can be selected to fully utilize the complementarity of vibrational spectra. Representative bands were selected for imaging, including benzene semicircular stretching at 1477cm ^-1 in infrared, benzene quadrant stretching at 1593cm ^-1 in Raman, C=O stretching at 1700cm ^-1 in infrared, and C≡C stretching at 2216cm ^-1 in Raman. Limited by the spectral selection rule, benzene semicircular stretching and C=O stretching are inactive in the Raman spectrum, while infrared is inactive in the alkyne band, as shown in Figure 16. Therefore, each single modality imaging itself cannot distinguish the mixture. The three compounds were successfully separated after spectral unmixing with unique vibrational activity patterns in the two modes, as shown in Figures 17 and 18. In addition, as shown in Figure 19, the present invention is superior to single-mode measurement in terms of specificity and selectivity as evaluated by the Pearson correlation coefficient (PCC).

Single-cell analysis is essential for tracking cell differentiation processes and studying their roles in disease models or tissue engineering for various clinical applications. Traditional phenotypic characterization methods are often time-consuming and invasive, resulting in limited temporal and spatial resolution. Therefore, a new technology is urgently needed to non-invasively monitor molecular dynamics during the initial stages of cell differentiation.

After verifying that the present invention has high spatial resolution and high specificity chemical imaging capabilities, in a specific embodiment of the present invention, in situ imaging and metabolic analysis were performed on mesenchymal stem cells (MSCs) as a control group and chondrocytes (CBs) as an experimental group after three days of differentiation. In order to further study the metabolic changes associated with lipid droplet synthesis, transmembrane state imaging with single beam detection under the infrared 1750cm ^-1 C=O band and Raman 2850cm ^-1 _CH2 symmetric stretching band was performed, as shown in Figure 20 and Figure 21 are two wave numbers to show the level of triglycerides (TAG) relative to total lipid droplets. As shown in Figure 22, a significant increase in the number of lipid droplets was observed, and the size distribution was wider, with a significant difference of p=0.007, indicating that lipid anabolism was significantly changed after differentiation induction. Considering the role of lipid droplets in antioxidant effects, the increased lipid droplets in the early stages of chondrocyte differentiation may indicate the activation of antioxidant pathways to alleviate the accumulation of reactive oxygen species.

The synthesis of complex large molecules, such as proteoglycans, is an important metabolic indicator of chondrocyte differentiation. Although the infrared 1080 cm ^-1 is considered to be the characteristic peak of proteoglycans in various infrared spectroscopy studies, the limitations of conventional infrared imaging in terms of spatial resolution and compatibility with cell imaging in aqueous environments have hindered the in situ detection of proteoglycan distribution. In order to effectively utilize the sensitivity of infrared and Raman, infrared 1080 cm ^-1 was used to depict proteoglycans, and Raman 2930 cm ^-1 of the _CH2 asymmetric stretching band was selected to represent the overall protein distribution, as shown in Figure 23. By performing ratio analysis on the transmembrane state image at 1080 cm ^-1 /2930 cm ^-1 , Figure 24 visualizes a large number of proteoglycan particles distributed in the cell body. Figure 25 quantifies the distribution of the size and relative intensity of proteoglycan particles, revealing that even in the early stages of differentiation induced for 3 days, there are significant differences in the synthesis of PD, i.e., p = 0.0011 for size distribution and p = 0.0012 for intensity distribution. PCC after differential analysis further highlights the higher specificity of the method provided by the present invention compared to single-modality measurements, as shown in Figure 26. In summary, these results indicate that the single-beam probed transmembrane state spectroscopy and imaging method provided by the present invention is a non-invasive and rapid single-cell analysis technique with high sensitivity, high specificity and high spatial resolution without any labeling or staining.

Identifying molecular features at the cellular level plays a key role in early disease detection and the study of pathological mechanisms. While omics-based technologies excel in screening disease-associated molecular markers, imaging-based methods provide valuable spatial information, enabling a deeper understanding of molecular mechanisms. However, traditional histopathological analysis requires prior knowledge and time-consuming preparations, such as immunofluorescence staining. In addition, there are considerable challenges in efficiently labeling most metabolites, including lipids and cholesterol, for in situ compositional analysis. The present invention addresses these challenges by providing label-free imaging with high molecular specificity, facilitating metabolic analysis under pathological conditions.

In a specific embodiment of the present invention, transmembrane state spectroscopy and imaging were performed in an Alzheimer's disease (AD) model, demonstrating the ability of in-situ high information content analysis. For the striatum of the brain, two sets of single-beam detection transmembrane state imaging were performed at infrared 1465cm ^-1 , Raman 2850cm ^-1 and infrared 1750cm ^-1 , Raman 2930cm ^-1 , as shown in Figure 27. The imaging results show that in the striatum, the fibrous structure is evenly distributed, and an obvious 2930cm ^-1 protein signal appears. It is worth noting that the presence of striatal corpuscles ranging in size from 20 to 100 microns was observed, which contained high levels of lipid and protein signals. With the ability of the present invention to have a higher detection sensitivity for small particles in the detection method, a large number of lipid droplets were visualized, and the infrared 1750cm ^-1 involving lipid C=O band and the infrared 1465cm ^-1 involving lipid _CH2 vibration and cholesterol methyl vibration were observed to be mainly distributed outside the striatum, and partially co-localized in the striatum.

To gain insight into the chemical composition, in situ IR and Raman spectra were simultaneously collected in different structures, including the fiber structure (S1), the co-localized droplets in the striatum (S2), and the droplets outside the striatum (S3), revealing their distinct compositions, as shown in Figure 28. Spectra s1 and s2 clearly show peaks at 1655 cm ^-1 (amide I), 1545 cm ^-1 (amide II), and 2930 cm ^-1 (CH ₂ asymmetric band), which are characteristic of the protein components in the fiber structure and the striatum. Notably, the peaks at 1750 cm ^-1 and 2850 cm ^-1 in spectrum s2 indicate a higher lipid content in the striatum compared to the fiber structure. In addition, the elevated cholesterol levels in the co-localized droplets were confirmed by the verification of multiple cholesterol characteristic peaks: IR 1375 cm ^-1 (CH ₃ symmetric bending), IR 1465 cm ^-1 (CH ₃ asymmetric bending), and Raman 2870 cm ^-1 (CH ₃ symmetric stretching). Spectrum S3 shows significant peaks at 1069 cm ^-1 and 1224 cm ^-1 , associated with POC and PO ₂ ^- vibrations, indicating a high phospholipid content. These results demonstrate the potential of complementary spectral imaging and in situ spectroscopic measurements to identify metabolic signatures of pathological conditions, which could aid disease detection and the development of targeted therapies.

In addition to single-cell analysis and metabolic imaging of tissue sections, it is also demonstrated that the transmembrane state imaging method provided by the present invention can become a versatile tool for studying microorganisms. C.elegans is a widely adopted model organism for mechanism studies related to aging and disease. Importantly, mechanistic studies of different molecules require at least dual wavenumber imaging. A single simultaneous dual wavenumber imaging was performed in C.elegans: infrared 1655cm ^-1 and Raman 2850cm ^-1 , corresponding to the protein amide I band and lipid _CH2 band, respectively, as shown in Figures 29 and 30, revealing the structure and biological composition of different organs inside C.elegans. Specifically, lipid components are mainly stored in the abdomen, which may be attributed to organs such as the gastrointestinal tract. In contrast, protein-rich areas are mainly located in the back and contain abundant body wall muscles.

The present invention realizes infrared and Raman imaging and spectroscopy detected by a single beam and detector, solves the problems of limited resolution of traditional infrared imaging and insufficient sensitivity of Raman imaging, and more importantly, makes it possible to simultaneously collect infrared and Raman images and perform more accurate quantitative biochemical analysis. It demonstrates the simultaneous infrared and Raman imaging of multiple chemical bonds with high resolution, high sensitivity and high accuracy in various application scenarios. It is worth noting that due to the complementary advantages of infrared and Raman vibrational modes and their inherent complementarity, the spectral and imaging methods provided by the present invention have enhanced specificity and selectivity, can realize quantitative analysis of complementary images, and are applicable to a wider range of scenarios. Dual-mode vibrational imaging inherits the richness of complementary spectroscopy in terms of structural and dynamic insights, and provides great opportunities and promotes molecular-level understanding for spectral imaging studies of complex biological and chemical systems such as in situ chemical reaction monitoring, single-cell metabolic analysis, pathological mechanism research and biological imaging. In addition, compared with complex multiplexed SRS techniques that are often limited to continuous spectral ranges, the method of the present invention has a wider spectral tunability and greatly reduces spectral redundant information. In addition, the concept of the method of the present invention can be extended to various spectral imaging modes for cross-modal imaging with high information dimensions. With the currently equipped picosecond laser source, the method of the present invention can become a versatile imaging and analysis platform compatible with a rich range of nonlinear optical imaging modalities, such as TA, TPEF, CARS, SHG, THG and SFG modalities, opening up new possibilities for optical imaging and sensing in biomedicine and materials science.

Limited by the range of laser wavelength, the infrared imaging band in this embodiment is limited to 900-2000 wavenumbers. By using other lasers, the imaging band using the photothermal effect in cross-modal spectrum and imaging of single-beam detection can be further expanded to ultraviolet light, visible light, near infrared to far infrared.

As used herein, the term "comprising" should be understood as inclusive and open, and should not be understood as exclusive. Specifically, when used in this specification including claims, the term "comprising" and its variations mean including specific features, steps or components. These terms are not to be interpreted as excluding the presence of other features, steps or components.

The specific implementation methods described above have described in detail the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The cross-mode spectrum and imaging method based on single beam detection is characterized by comprising the following steps of:

1) Regulating and controlling a beam of stimulated radiation light and a beam of pump light with tunable wavelength to enable the two beams to coincide in a time domain and a space domain, regulating a beam of middle infrared light with tunable wavelength to enable a light path of the middle infrared light to be collinear with the stimulated radiation light and the pump light, and further focusing three beams of light to enable focuses of the three beams of light to coincide in space;

2) Setting a target sample to be detected on a focus of three focused beams of light;

3) The wavelength of the pumping light is adjusted, so that the energy difference between the pumping light and the stimulated radiation light is matched with the Raman vibration level of the target molecule, the stimulated Raman scattering effect is realized, the wavelength of the mid-infrared light is adjusted, so that the mid-infrared light is matched with the infrared vibration level of the target molecule, the local photo-thermal relaxation effect around the target molecule is realized, and the target molecule in the three light focuses simultaneously generates the stimulated Raman scattering effect and the mid-infrared thermal relaxation effect generated by mid-infrared absorption; so that the stimulated Raman scattering signal generated by the stimulated Raman scattering effect and the mid-infrared light thermal signal generated by the mid-infrared light thermal relaxation effect are carried in the light intensity of the pumping light;

4) Modulating the light intensity of stimulated radiation light and mid-infrared light on a time domain by utilizing the time characteristic difference of the stimulated Raman scattering effect and the photo-thermal relaxation effect, so that signals of the stimulated Raman scattering effect and the photo-thermal relaxation effect are in different frequency intervals;

5) After stimulated Raman scattering signals and mid-infrared light thermal signals generated in different frequency intervals are carried by pumping light, the pumping light is used as detection light to be input into a photoelectric detector to be converted into a photovoltage signal, and a frequency domain filtering technology is adopted to separate the stimulated Raman scattering signals and the mid-infrared light thermal signals from the photovoltage signal;

6) Scanning the spatial position of a target sample, and simultaneously collecting stimulated Raman scattering signals and mid-infrared light thermal signals to realize the collection of cross-mode imaging data, wherein the collected cross-mode imaging data generates an imaging chart through spectrum and imaging processing equipment;

7) And scanning the wavelength of the pumping light and the wavelength of the mid-infrared light, and carrying out spectrum acquisition on molecules in the common focus of the stimulated radiation light, the pumping light and the mid-infrared light to obtain spectrums of all modes.

2. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 1), the stimulated radiation light and the pump light are ultrafast lasers, the pulse width is in the order of femtosecond to picosecond, and the wavelength range is from visible light to near infrared light wave band; the wavelength of the pump light is shorter than that of the stimulated radiation light, and the photon energy difference between the stimulated radiation light and the pump light covers the molecular vibration spectrum range.

3. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 1), the stimulated radiation light and the pump light need to be adjusted to coincide in a time domain; the mid-infrared light does not form time domain coincidence with the stimulated radiation light and the pump light, so as to avoid nonlinear effect with the stimulated radiation light and the pump light.

4. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 3), the stimulated raman scattering effect reaches the highest signal value when the time delay of the stimulated radiation light and the pump light is 0 picoseconds, and the detection of the time resolution coherent raman scattering is realized by adjusting the time delay of the stimulated radiation light and the pump light pulse; the stimulated Raman scattering effect is expressed in the form of stimulated Raman gain on the intensity of the stimulated radiation light, and is expressed in the form of stimulated Raman loss on the intensity of the pump light.

5. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 3), the mid-infrared light thermal relaxation is an energy non-radiation relaxation process of the target molecule local area, and the stimulated radiation light and the pump light irradiated on the target molecule are simultaneously subjected to photo-thermal lens phenomenon, and the light intensity of the stimulated radiation light and the pump light is modulated.

6. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 4), the time characteristic difference between the stimulated raman scattering effect and the photo-thermal relaxation effect is specifically: the time scale of the stimulated Raman scattering effect is hundred femtoseconds, the time scale of the photo-thermal relaxation effect is in the range of nanoseconds to microseconds, and the response time is 4 to 7 orders of magnitude different; the time characteristic difference of the stimulated Raman scattering effect and the photo-thermal relaxation effect is utilized to respectively modulate the stimulated radiation light and the mid-infrared light, so that the stimulated Raman effect is modulated within a range of one megahertz to hundred kilohertz, the photo-thermal relaxation effect is modulated within a range of one thousand to hundred kilohertz, and zero crosstalk detection of the cross-modal spectrum and the imaging signal is realized.

7. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 4), the modulation of the stimulated radiation light is realized through an electro-optic modulator or an acousto-optic modulator, and the modulation of the mid-infrared light is realized through electric modulation or chopper modulation.

8. The cross-modality spectroscopy and imaging method of claim 1, wherein: the pumping light carrying the stimulated Raman scattering signal and the mid-infrared light thermal signal is formed by stimulated Raman modulation and mid-infrared light thermal modulation in a frequency domain, the pumping light is used as the detection light to realize optimal spatial resolution and detection sensitivity, the pumping light used as the detection light is collected into the photoelectric detector through the forward direction or the backward direction, and the forward direction is the incidence direction of the pumping light, and the backward direction is the opposite direction of the incidence direction of the pumping light.

9. The cross-modality spectroscopy and imaging method of claim 1, wherein: in the step 5), the frequency domain filtering technology converts the photovoltage signal acquired by the photoelectric detector from the time domain to the frequency domain, and the stimulated raman scattering signal and the mid-infrared thermal signal are extracted, separated and amplified by utilizing the frequency spectrum characteristics of the signal.

10. A cross-modality spectroscopy and imaging apparatus for implementing the cross-modality spectroscopy and imaging method of claim 1, comprising:

the light source module comprises a first light source for emitting stimulated radiation light and pump light, a second light source for emitting mid-infrared light, an electro-optical modulator for performing time domain modulation on the stimulated radiation light, and a light path module for performing spatial domain superposition adjustment and mid-infrared light collinearly adjustment on the stimulated radiation light and the pump light;

The microscope module comprises a microscope frame, a lens module for adjusting light, an infrared focusing lens for focusing middle infrared light and collecting pumping light and a carrying sample scanning table for carrying samples;

the detection module comprises a collection light path for collecting signals, a filter set for filtering out pumping light, a photoelectric detector for converting the intensity of the pumping light into a photovoltage signal and a preamplifier;

The spectrum and imaging acquisition module comprises a data acquisition card, a filter device and spectrum and imaging processing equipment; the data acquisition card and the filter device are used for acquiring cross-mode imaging data and spectra; the spectrum and imaging processing equipment is used for controlling the data acquisition card, the object carrying sample table, the first light source, the second light source and the filter device.