CN118010700A

CN118010700A - Cross-modal spectrum and imaging method and device based on single-beam detection

Info

Publication number: CN118010700A
Application number: CN202410016092.6A
Authority: CN
Inventors: 张德龙; 李炫祯; 傅鹏程; 张永庆
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2024-01-05
Filing date: 2024-01-05
Publication date: 2024-05-10

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 spectrum and imaging method and device based on single-beam detection

Technical Field

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

Background

The goal of cross-modal imaging is to provide unique and complementary insights into various targets by synergistically combining different microscopic imaging modalities, providing an overall view of molecular dynamics for comprehensive chemical, physical and biological analysis. The cross-modality imaging may provide additional molecular spatial information compared to a single imaging method. For example, the introduction of fluorescence imaging modalities to complement the lack of specificity of digital holography and interferometric scatter imaging demonstrates the ability of cross-modality imaging to study organelle interactions and nanoscale dynamic event tracking. In face of the challenges of labeling small molecules and metabolites, label-free imaging methods have been developed to supplement molecular species limitations in fluorescence imaging modalities by capturing intrinsic molecular structure 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, providing new insight for the study of cellular metabolism and pathological mechanisms. In addition, by combining multiple imaging modes such as scattering, optoacoustic, phase and fluorescence, the pump detection technology based on the photo-thermal effect induced by light absorption shows a remarkable synergistic effect, and the resolution, sensitivity and flux of label-free vibration imaging are greatly improved.

For non-invasive detection of intrinsic fingerprint information of molecular vibrations, infrared and raman spectra have found plentiful applications in various fields, especially infrared spectra, whose absorption cross section (about 10 ^-18cm²) is much higher than that of conventional raman spectra (10 ^-28cm²). However, it is difficult to determine complete molecular structure information with a single spectral modality, because vibrational modes with raman activity do not necessarily have infrared activity and vice versa, infrared spectra are very sensitive to asymmetric vibrations, while raman spectra are sensitive to symmetric vibrations, which results in incomplete spectral information. Thus, simultaneous detection in combination with infrared and raman spectra is necessary to provide complementary and comprehensive vibrational spectral information to understand more deeply the complex process. For example, by integrating infrared and raman spectroscopic measurements, it has been demonstrated in the chemical arts to be able to identify new NO _x adsorbed state species and to visualize information related to the location of NO _x storage efficiency in heterogeneous catalytic processes. Recent research on CO reduction reactions has utilized in situ enhanced infrared and raman spectroscopy techniques to reveal the mechanism of formation during adsorption of CO subpopulations. Early attempts to image biological samples using sequential infrared and raman spectroscopy have shown their potential in providing a full spectrum of cell composition and enhanced biomarker identification.

However, it is not feasible to directly combine infrared and raman modes because of the significant differences between their different physical properties (absorption and scattering) and spectral windows (mid-infrared and visible light). Infrared and raman combined detection schemes have been proposed. For example IRaman combines mid-infrared thermography with single point raman spectroscopy. To overcome the conflict of infrared and raman mode selection rules, a nonlinear optical process is introduced, including two-photon excited raman spectroscopy, i.e., super raman spectroscopy. In addition, single point spectroscopic measurements of complementary vibrational spectra are achieved through fourier transform coherent anti-stokes raman scattering (raman sensitive) and intra-pulse difference frequency generation (infrared sensitive) processes. The combination of coherent anti-stokes raman scattering and three-photon processes generated by third-order sum frequency improves infrared and raman imaging resolution. However, developing simultaneous infrared and raman imaging platforms with high resolution and quantitative chemical imaging functionality remains a significant challenge.

In view of the above, there is still a lack of a solution for simultaneous spectroscopic and imaging of infrared and raman modalities, and no effective solution has been found. This problem requires further research and development to meet the spectral and imaging requirements of the optical imaging field for simultaneous realization of infrared and raman modes.

Disclosure of Invention

Based on the technical bottleneck, the invention provides a cross-mode spectrum and imaging method based on single-beam detection, which can realize simultaneous infrared and Raman spectrum and imaging acquisition in a label-free, high-sensitivity and high-spectrum range.

The invention firstly provides a cross-mode spectrum and imaging method based on single beam detection, which comprises the following steps:

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.

In the step 1), the stimulated radiation light and the pumping 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; 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 coincide in the time domain; the pulsed mid-infrared light does not form a time domain coincidence with the stimulated radiation light and the pump light, so as to avoid nonlinear effects with the stimulated radiation light and the pump light.

The invention also provides a cross-modal spectrum and imaging device for realizing the cross-modal spectrum and imaging method, which comprises the following steps:

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.

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

Compared with the prior art, the invention has the beneficial effects that at least the following steps are included:

The time domain modulation is carried out by utilizing time scale difference characteristics of different physical processes, so that 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 large difference of optical properties and different detection schemes is fundamentally solved, and simultaneous imaging of infrared and Raman complementary spectrum modes which are difficult to realize is realized.

The invention not only gets rid of the limitations of low resolution, water absorption, weak signals of Raman spectrum and the like of infrared imaging, but also enables photo-thermal imaging and stimulated Raman scattering imaging to detect on the same detection light, thereby greatly improving the resolution, sensitivity and speed of infrared imaging.

And thirdly, the invention uses a single detection beam detection method and a single detector detection method, simplifies the system complexity, avoids the crosstalk problem of the traditional multi-mode imaging, and realizes the crosstalk-free imaging. And the imaging flux is improved, and the imaging capability of the cross-spectrum mode is realized.

The method realizes simultaneous imaging of different physical processes from picoseconds to microseconds, has the characteristics of mutual compatibility and universality, and is basically suitable for any multi-mode imaging method based on time domain modulation.

Fifth, the present invention is generally applicable to all molecules and is not limited to mid-infrared absorption. The invention is applicable to any imaging modality based on absorption detection, which benefits from the scheme of photo-thermal detection; and provides more spectrum windows for various imaging modes excited by picosecond ultrafast laser, and can be compatible with various ultrafast optical modes, such as two-photon fluorescence, transient absorption, coherent anti-Stokes Raman imaging, second harmonic imaging, third harmonic imaging and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the energy levels of a single beam detection across the modal spectrum and imaging;

FIG. 2 is a schematic diagram of an experimental light path portion according to an embodiment of the present invention;

FIG. 3 is a signal transmission and system control diagram according to an embodiment of the present invention;

FIG. 4 is a waveform diagram of an incident laser pulse including pump light, stimulated emission light, and mid-IR light pulses in the time domain according to an embodiment of the present invention;

FIG. 5 is a diagram showing the intensity of the detection light in the time domain according to the embodiment of the present invention;

FIG. 6 is a diagram showing the intensity of the detection light in the frequency domain according to the embodiment of the present invention;

FIG. 7 is a graph of a spectrum of oil film detection in accordance with an embodiment of the present invention;

FIG. 8 is a graph of infrared and Raman spectrum data on 500nm PMMA microspheres measured simultaneously in accordance with an embodiment of the present invention;

FIG. 9 is an infrared and Raman imaging diagram of 500nm PMMA plastic microspheres collected simultaneously in an embodiment of the invention;

FIG. 10 is a plot of the cross-section of the infrared and Raman signals and the Gaussian fit of a single microsphere indicated by the arrows in FIG. 9;

FIG. 11 is a three-dimensional infrared imaging of a single 500nm PMMA plastic microsphere collected by an embodiment of the present invention;

FIG. 12 is a three-dimensional Raman imaging diagram of a single 500nm PMMA plastic microsphere acquired simultaneously with FIG. 11 according to an embodiment of the present invention;

FIG. 13 is a three-dimensional reconstruction of infrared and Raman from the single 500nm PMMA plastic microspheres of FIGS. 11 and 12;

FIG. 14 is a longitudinal cross-section of the three-dimensional imaging of the single 500nm PMMA plastic microsphere of FIGS. 11 and 12 and a Gaussian fit result;

FIG. 15 is an infrared transmission and spontaneous Raman spectrum of Palmitic Acid (PA), 1, 4-diphenyldiacetylene (DiPhDY) and Triphenylphosphine (TPP) used in the examples of the invention;

FIG. 16 shows the IR and Raman activities at different wavenumbers for three chemicals used in the examples of the present invention;

FIG. 17 is an infrared and Raman imaging diagram of a mixture of three chemical substances simultaneously collected in equal proportions according to an embodiment of the invention;

FIG. 18 is an overlay of a pure material profile and three material profiles separated from the data of FIG. 17;

fig. 19 shows a schematic diagram of pearson correlation coefficients between the imaging maps shown in fig. 17;

FIG. 20 is an image of control and experimental group cells acquired simultaneously in accordance with an embodiment of the present invention at 1750cm ^-1 and Raman 2850cm ^-1 infrared;

FIG. 21 is a ratio imaging plot of control and experimental cells at infrared 1750cm ^-1 and Raman 2850cm ^-1;

FIG. 22 is a distribution histogram of the number of lipid droplets in cells of the control and experimental groups as a function of relative intensity;

FIG. 23 is an image of control and experimental group cells acquired simultaneously in accordance with an embodiment of the present invention at 1080cm ^-1 IR and 2930cm ^-1 Raman;

FIG. 24 is a ratio imaging of control and experimental cells at 1080cm ^-1 and 2850cm ^-1 infrared and their partial magnified images;

FIG. 25 is a graph showing the distribution of the area and relative intensity of the polysaccharide protein particles in the cells of the control and experimental groups;

Fig. 26 shows a diagram of pearson correlation coefficients between the imaging plots of the control and experimental group cells shown in fig. 20 and 21;

FIG. 27 shows simultaneous dorsal infrared and anterior Raman imaging of striatum in mouse brain acquired in accordance with an embodiment of the invention;

FIG. 28 is an in situ infrared and Raman spectrum plot from three locations of interest indicated by arrows in FIG. 27;

FIG. 29 shows an infrared imaging of caenorhabditis elegans collected in an embodiment of the invention;

fig. 30 shows a raman image of caenorhabditis elegans collected according to an embodiment of the invention.

Detailed Description

In general, embodiments described herein relate to obtaining multispectral images using frequency multiplexing techniques to enable single-beam detection of cross-modal spectral information. As required, embodiments of the present invention are disclosed herein. It should be noted, however, that the implementation methods referred to herein are merely exemplary, so the present invention may be embodied in a variety of different and alternative forms.

Moreover, the figures shown herein are partially schematic illustrations of the drawings that are not necessarily to scale and some features may be exaggerated or minimized to show details of particular matters in order to highlight novel aspects of the present invention and to allow for simplicity of drawing. Also in the drawings herein, particularly the device drawings, related elements may have been omitted to prevent obscuring many innovative aspects of the new device. Therefore, specific details of the methods, structures, and functions disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention. For purposes of introduction and not limitation, disclosed herein are methods for obtaining multispectral images using frequency multiplexing techniques to enable single-beam detection of cross-modal spectral information.

The detection principle of the invention can be illustrated by an energy level diagram, and the molecules with specific structures have Raman vibration energy level and infrared vibration energy level, as shown in figure 1. A beam of pumping light and a beam of stimulated radiation light are introduced into specific molecules to irradiate, the molecules are excited to a virtual energy level by the pumping light, when the energy difference between the pumping light and the stimulated radiation light is matched to the energy of Raman vibration, the introduced stimulated radiation light can generate stimulated radiation to Raman photons, a Raman signal is amplified by about 10 ⁸ times, and the amplified Raman signal is called stimulated Raman signal: the Stimulated Raman Loss (SRL) signal is carried on the light intensity of the pump light, and the Stimulated Raman Gain (SRG) signal is carried on the light intensity of the stimulated radiation light; the molecule is irradiated by a beam of mid-infrared light, but the energy of the mid-infrared light is matched with the energy of the infrared vibration energy level, the molecule can directly absorb the infrared energy, the absorbed energy can cause the ambient temperature to rise through dissipation, thereby changing the local refractive index and generating deformation, at the moment, obvious photo-thermal effect is generated, the propagation of the light beam irradiated at the same space position can be modulated by a photo-thermal lens, which is called mid-infrared photo-MIP signal, and the infrared spectrum is linearly related to the MIP signal intensity.

It is therefore reasonable to propose a pump-probe scheme to detect the idea of both processes simultaneously. When the above-mentioned three beams of light are simultaneously irradiated on the sample, stimulated raman scattering signals and photothermal signals are simultaneously generated on the light intensities of the pump light and the stimulated radiation light. Such a detection method can bring two distinct modalities into the same dimension for detection.

The stimulated raman process and the photothermal process mentioned above, as shown in fig. 1, occur in the case where the pump light, the stimulated radiation light, and the mid-infrared light are continuous light and pulsed light. The invention utilizes the distinct characteristics of stimulated Raman process and photothermal process on time scale: the stimulated Raman scattering process occurs in a time scale range from femtosecond to picosecond, the photothermal relaxation process occurs in a process from nanosecond to microsecond, a pulse light excitation detection scheme is adopted, and modulation is respectively carried out in respective time windows of the stimulated Raman scattering process and the photothermal relaxation process, so that the minimum signal crosstalk and the highest modulation efficiency are realized, and the single-beam detection cross-modal spectrum imaging is realized.

In addition, the pump light and the stimulated radiation light can carry stimulated Raman scattering signals and mid-infrared light thermal signals, and can be used as detection light to realize single-beam detection of cross-mode spectrum imaging. In consideration of various factors, such as the detection quantum efficiency and the spot diffraction limit of a conventional silicon-based detector, the invention selects pump light as detection light to realize better performance, including higher detection sensitivity and higher imaging resolution.

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

1) The first light source 1 is an ultrafast laser light source, emits two beams of laser light, the pulse width is 2 picoseconds, the basic pulse repetition frequency is 80 megahertz, one beam is pump light with tunable wavelength, the tunable range is 700-990 nanometers, and the two beams of laser light pass through the first broad beam lens 2, the second broad beam lens 3 is adjusted to be parallel light, and then propagate through the dichroic mirror 10. The other beam is stimulated radiation light with the wavelength fixed at 1031 nanometers, the stimulated radiation light is collimated and adjusted to be parallel light by a third broad beam lens 4 and a fourth broad beam lens 5 respectively, and then is guided to an electro-optical modulator 9 through a first reflecting mirror 6, a second reflecting mirror 7 and a third reflecting mirror 8 in sequence, modulated to be 20 MHz, reflected by a bicolor mirror, and combined with pumping light by adjusting the angles of the three reflecting mirrors.

2) The distance of the third reflecting mirror 8 is used for adjusting the optical path of the stimulated radiation by moving the second reflecting mirror 7 so as to adjust the time coincidence of the pump light and the stimulated radiation light pulse focusing on the sample; the moving direction is pointed out by an arrow, 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 can be realized by adjusting the time delay of the stimulated radiation light and the pump light pulse.

3) The pump light and the stimulated radiation light after beam combination are guided by the reflector 11, transmitted through the 50-50 beam splitter 12 and focused on a sample by the refraction type objective lens 18; the wavelength-tunable mid-infrared light emitted by the second light source 13, the wavelength-tunable range is 5-10.5 micrometers, the repetition frequency is adjusted to 100 kilohertz by electrically modulating the laser, the pulse width is 500 nanoseconds, and then the mid-infrared light is guided by the fifth reflecting mirror 14 and passes through the beam splitter 15;

4) The mid infrared light then passes through the sixth mirror 16, and the seventh mirror 17 is directed to the infrared focusing mirror 19 and focused on the sample. The optical axis of the middle infrared light path is completely overlapped with the optical path optical axes of the pump light and the stimulated radiation light by adjusting the included angle of the two reflectors and the space position of the infrared focusing mirror, and the focal spot focused by the middle infrared laser is overlapped with the focal spot of the pump light and the stimulated radiation light in space;

5) The sample is carried on the carrying sample scanning table 20, the wavelength of the pumping light is tuned to enable the energy difference of the pumping light and the stimulated radiation light to be matched with the corresponding Raman vibration energy level, the wavelength of the mid-infrared light is tuned to the infrared vibration energy level, the sample generates corresponding stimulated Raman scattering signals and mid-infrared thermal signals, and the signals are carried in the pumping light and the stimulated radiation light;

6) The pump light carrying the stimulated Raman scattering signal and the mid-infrared thermal signal is used as detection light, and can be detected through backward or forward. The forward direction is the incidence direction of the pump light, and the backward direction is the opposite direction to the incidence direction of the pump light. The backward detection flow is as follows; after being collected by the refraction type objective lens 18, the light is reflected by the 50-50 beam splitter, filtered by the first filter set 21 and then enters the first photoelectric detector 22; the forward detection flow is as follows: the light is collected forward through an infrared focusing mirror 19, guided by a seventh reflecting mirror 17 and a sixth reflecting mirror 16 in sequence, reflected by a beam combining mirror 15 and separated from a light path, filtered by a second optical filter set 23, and then incident on a second photoelectric detector 24; when forward detection is performed, an infrared focusing mirror for collecting pump light needs to maintain high transmittance and collection efficiency for pump light to reduce loss of pump light at the time of detection.

7) The photodetectors 22, 24 convert the obtained optical signals into electrical signals, input the electrical signals to a preamplifier 28 for further voltage signal amplification, then input the electrical signals to a filter device 26 for digital-to-analog conversion, and the filter device receives the modulated frequency signals of the second light source 13 and the modulated frequency signals of the electro-optical modulator 9 as central frequency bands of filtering, and performs narrow-band filtering at 100 khz and 20 mhz simultaneously to separate out signal components, namely, simultaneously detects stimulated raman scattering signals and mid-infrared optical thermal signals;

8) The object sample stage 20 is used for carrying out space scanning and simultaneously collecting output signals of a filter device, so that cross-mode infrared and Raman imaging data collection based on single-beam detection is realized, and the collected data are subjected to pixel segmentation and averaging through the spectrum and imaging processing equipment 25 to form an imaging chart; the imaging speed, field of view, pixel size and acquisition time are controlled by the spectroscopic 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 pumping light emitted by the first light source 1, spectrum acquisition is performed on molecules in a common focus of the stimulated radiation light, the pumping light and the mid-infrared light, so that cross-mode in-situ infrared and Raman spectrum data acquisition based on single-beam detection is realized, and cross-mode in-situ infrared and Raman spectrum acquisition wave bands and speeds are controlled by a spectrum and imaging processing device 25;

In order to facilitate understanding of the detection scheme of the present invention, the pulse sequence diagram of the incident pump light, the stimulated radiation light and the mid-infrared light shown in fig. 4 is combined, wherein f _P,f_S,f_M represents the modulation frequencies of the pump light, the stimulated radiation light and the mid-infrared light, respectively. In one embodiment of the invention, f _P,f_S,f_M is 80 MHz, 20 MHz, 100 kHz, respectively. The pulse width is 2 picoseconds, and the propagation of the pump light with the tunable wavelength and the repetition frequency of 80 MHz generates photo-thermal modulation. The pump light intensity with the repetition frequency of 80 MHz and tunable wavelength is encoded with a set of stimulated Raman loss signals with the modulation frequency of 20 MHz at high frequency and a set of photothermal signals with the modulation frequency of 100 kHz at low frequency. The pumping light intensity is converted into a photovoltage signal through a photoelectric detector, and the photovoltage signal is input to a filter device after being amplified in advance. In order to more conveniently understand the signal composition generated by the embodiment, the characteristics of the waveform diagram of the probe light in the time domain and the frequency domain are described with reference to fig. 5 and 6, and the pump light is selected as the probe light to realize better performance. Fig. 5 shows a time domain pump waveform, which carries an SRL signal and a MIP signal. As shown in fig. 6, the frequency domain component of the detected light intensity in fig. 5 is shown, the carried SRL signal is in the form of a peak at frequency f _S, and the carried MIP signal is in the form of a series of harmonic peaks with fundamental frequency f _M. In this embodiment, the distance between the infrared light modulation frequency and the stimulated radiation light modulation frequency in the frequency spectrum is f _S-f_M, which is 19.9 mhz, so that the mixing of the MIP signal and the SRS signal can be avoided. And the high-frequency modulation can avoid low-frequency noise and obviously improve the signal-to-noise ratio while avoiding signal crosstalk. Fig. 7 shows the frequency domain composition of the signal actually measured on the olive oil sample, no frequency components are observed at f _S±f_M, which indicates that cross-modal spectrum of single beam detection and imaging method there is no crosstalk between signals.

In one embodiment of the present invention, a single beam detection cross-modal spectrum and imaging method has been experimentally performed to collect cross-modal spectrum data and imaging data for 500 nm polymethyl methacrylate PMMA plastic microspheres. Fig. 8 shows infrared and raman spectral data. Using the collected spectral data, a c=o telescopic vibration band with infrared mode 1730cm ^-1 was selected, a CH ₃ asymmetric telescopic vibration band with raman mode 2957cm ^-1, and cross-mode imaging with single beam detection was performed, the result is shown in fig. 9. FIG. 10 shows a cross-sectional view of the invention for imaging 500 nm beads, from which the resolution of infrared and Raman imaging can be calculated by deconvolution. The resolution of the infrared imaging is 599 nanometers, the resolution of the Raman imaging is 490 nanometers, and the resolution of the Raman imaging is slightly superior to the infrared imaging mainly because the Raman imaging is based on a nonlinear stimulated Raman scattering scheme, the resolution is determined by the pump light and the stimulated radiation light together, and the resolution of the infrared imaging is mainly determined by the pump light.

And the single microsphere is subjected to single-beam detection transmembrane three-dimensional imaging, as shown in fig. 11 and 12, which are respectively images acquired under infrared 1730cm ^-1 and raman 2957cm ^-1, and the three-dimensional reconstruction is shown in fig. 13. Fig. 14 is an axial cross-sectional view of fig. 13. Through three-dimensional imaging, the imaging diagrams of the two modes are free from focal plane deviation, so that the reliability of the transmembrane imaging system provided by the invention is further ensured, and the feasibility of quantitatively analyzing the transmembrane data is further ensured.

To demonstrate the enhanced chemical specificity of the present invention, in one embodiment of the present invention, single beam detection transmembrane state imaging of three mixtures comprising PA palmitate, 1, 4-diphenyldiacetylene DiPhDY and triphenylphosphine TPP was performed, comprising a pattern of similar structural and overlapping spectral bands as shown in figure 15. Because the invention has wide and independent infrared and Raman spectrum coverage, chemical bands with higher specificity can be selected to fully utilize the complementarity of vibration spectrum. Representative bands were selected for imaging, including benzene semicircle stretching of infrared 1477cm ^-1, benzene quadrant stretching of raman 1593cm ^-1, c=o stretching of infrared 1700cm ^-1, and c≡c stretching of raman 2216cm ^-1. Limited by the rule of spectrum selection, benzene semicircle stretching and c=o stretching are inactive in raman spectrum, while infrared appears inactive in alkyne band, as shown in fig. 16. Thus, each single modality imaging cannot distinguish the mixture itself. The three compounds were successfully separated after spectral unmixing, as shown in fig. 17 and 18. Furthermore, the present invention is superior to single modality measurements in terms of specificity and selectivity as shown in fig. 19, as assessed by Pearson Correlation Coefficient (PCC).

Single cell analysis is critical for tracking the cell differentiation process and studying its role 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. Thus, there is an urgent need for a new technique that can non-invasively monitor molecular dynamics at the initial stage of cell differentiation.

After verifying the chemical imaging capability of the present invention with high spatial resolution and high specificity, in one embodiment of the present invention, in situ imaging and metabolic analysis was performed on Mesenchymal Stem Cells (MSCs) as a control group and Chondroblasts (CBs) as an experimental group after three days of differentiation. To further investigate metabolic changes associated with lipid droplet synthesis, single beam detection transmembrane imaging under infrared 1750cm ^-1 c=o band and raman 2850cm ^-1CH₂ symmetric telescopic band was performed, as shown in fig. 20, fig. 21 for two wavenumbers, to reveal Triglyceride (TAG) levels relative to total lipid droplets. As shown in fig. 22, a significant increase in lipid droplet number was observed and the size distribution was broader with a significant difference, p=0.007, indicating a significant change in lipid anabolism after differentiation induction. Given the role of lipid droplets in antioxidant effect, increased lipid droplets at the early stages of chondrocyte differentiation may indicate that the antioxidant pathway is activated to mitigate 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 1080cm ^-1 infrared is considered a characteristic peak of proteoglycans in various infrared spectroscopy studies, conventional infrared imaging has limitations in terms of spatial resolution and compatibility with cell imaging in aqueous environments, preventing in situ detection of proteoglycan distribution. To effectively use the sensitivity of infrared and raman, proteoglycans were depicted using the infrared 1080cm ^-1 and raman 2930cm ^-1 of the CH ₂ asymmetric stretch band was chosen to represent the overall protein distribution, as shown in figure 23. By ratio analysis of transmembrane state images at 1080cm ^-1/2930cm^-1, figure 24 visualizes the large number of proteoglycan particles distributed in the cell body. Fig. 25 quantifies the distribution of proteoglycan particle size and relative intensity, revealing that there was a significant difference in PD synthesis even at the early stage of 3 days of induced differentiation, i.e., size distribution p=0.0011, intensity distribution p=0.0012. The PCC after differential analysis further highlights that the method provided by the present invention provides higher specificity compared to single mode measurement, as shown in fig. 26. Taken together, these results demonstrate that the single beam detected transmembrane state spectra provided by the present invention have high sensitivity, high specificity and high spatial resolution as a non-invasive, rapid single cell analysis technique in imaging methods without the need for any labeling or staining.

Recognition of molecular features at the cellular level plays a key role in early disease detection and in the study of pathological mechanisms. Although histology-based techniques perform well in screening for disease-related molecular markers, imaging-based methods provide valuable spatial information to allow for a deeper understanding of molecular mechanisms. However, traditional histopathological analysis requires a priori knowledge and time-consuming preparation work, such as immunofluorescent staining. Furthermore, labeling most metabolites, including lipids and cholesterol, efficiently for in situ compositional analysis presents considerable challenges. The present invention addresses these challenges by providing polymer-specific label-free imaging, which facilitates metabolic analysis under pathological conditions.

In one embodiment of the invention, transmembrane state spectroscopy and imaging are performed in an Alzheimer's Disease (AD) model, demonstrating the ability to analyze high information content in situ. For the striatum of the brain, two sets of single beam detection transmembrane imaging were performed at infrared 1465cm ^-1, raman 2850cm ^-1, and infrared 1750cm ^-1, raman 2930cm ^-1, respectively, as shown in fig. 27. The imaging results showed that the fibrous structures were uniformly distributed within the striatum, and a distinct 2930cm ^-1 protein signal appeared. Notably, the presence of striatal bodies of varying sizes from 20 to 100 microns was observed, which contained high levels of lipid and protein signaling. With the ability of the invention to have a higher detection sensitivity for small particles in the detection method, a large number of lipid droplets were visualized, with a major distribution outside the striatum observed at infrared 1750cm ^-1 involving the lipid c=o band and infrared 1465cm ^-1 involving the lipid CH ₂ vibration and the cholesterol methyl vibration, and partially co-localized within the striatum.

In order to gain an in-situ understanding of the chemical composition, both in-situ infrared and raman spectra were acquired in different structures, including fibrous structure (S1), co-localized droplets in the striatum (S2), and droplets outside the striatum (S3), revealing their distinct compositions, as shown in fig. 28. Spectra s1 and s2 clearly show peaks at 1655cm ^-1 (amide I), 1545cm ^-1 (amide II) and 2930cm ^-1(CH₂ asymmetric bands), which are characteristic of the fibrous structure and protein components in the striatum. Notably, peaks 1750cm ^-1 and 2850cm ^-1 in spectrum s2 indicate a higher lipid content in the striatum compared to the fibrous structure. In addition, by verification of multiple cholesterol characteristic peaks: infrared 1375cm ^-1(CH₃ symmetrically curved), infrared 1465cm ^-1(CH₃ asymmetrically curved), and raman 2870cm ^-1(CH₃ symmetrically stretched), demonstrating an increase in cholesterol levels in co-localized droplets. The spectrum s3 shows significant peaks at 1069cm ^-1 and 1224cm ^-1, associated with P-O-C and PO ₂ ^- vibrations, indicating high phospholipid content. These results demonstrate the potential of complementary spectral imaging and in situ spectral measurements in identifying metabolic features of pathological conditions, which are helpful for disease detection and development of targeted therapies.

Besides single cell analysis and metabolic imaging of tissue sections, the transmembrane imaging method provided by the invention is also shown to be a multifunctional tool for researching microorganisms. Elegans is a widely used model organism for the study of mechanisms associated with aging and disease. Importantly, the mechanical investigation of different molecules requires at least dual-wave imaging. Simultaneous double wave number imaging was performed in C.elegans: infrared 1655cm ^-1 and raman 2850cm ^-1, corresponding to the protein amide I band and lipid CH ₂ band, respectively, as shown in fig. 29 and 30, revealed the structure and biological composition of the different organs inside c.elegans. In particular, the lipid component is mainly stored in the abdomen, possibly due to organs such as the stomach and intestine. In contrast, the protein-rich region is located mainly on the back, containing rich body wall muscles.

The invention realizes infrared and Raman imaging and spectrum detected by a single beam and a detector, solves the problems of limited resolution and insufficient Raman imaging sensitivity of the traditional infrared imaging, and more importantly, enables the simultaneous acquisition of infrared and Raman imaging and the more accurate quantitative biochemical analysis to be possible. Infrared and raman simultaneous imaging of multiple chemical bonds that achieve high resolution, high sensitivity and high accuracy under various application scenarios is demonstrated. Notably, due to the infrared and raman vibration modes and the inherent complementarity advantages thereof, the spectrum and imaging method provided by the invention have enhanced specificity and selectivity, can realize quantitative analysis of complementary images, and are suitable for wider scenes. The dual-mode vibration imaging inherits the richness of complementary spectroscopy in terms of structure and dynamic insight, and provides great opportunities and facilitates understanding of molecular level for in-situ chemical reaction monitoring, single-cell metabolic analysis, pathological mechanism research, biological imaging and other spectral imaging research of complex biological and chemical systems. In addition, the method of the present invention has broader spectral tunability and greatly reduces spectrally redundant information compared to complex multiplexed SRS techniques that tend to be limited to a continuous spectral range. Furthermore, the concept of the method of the present invention can be extended to various spectral imaging modes for cross-modality imaging of high information dimension. By means of the picosecond laser source equipped at present, the method can become a multifunctional imaging and analysis platform compatible with rich nonlinear optical imaging modes, such as TA, TPEF, CARS, SHG, THG and SFG modes and the like, and opens up new possibilities for optical imaging and sensing of biomedicine and material science.

The wavelength range of the laser is limited, the band of infrared imaging is limited to 900-2000 wave numbers in the embodiment, and other lasers are utilized to further expand the cross-mode spectrum detected by a single beam and the imaging band utilizing the photo-thermal effect in imaging, and the infrared imaging band can be expanded to ultraviolet light, visible light and near infrared to far infrared.

As used herein, the term "comprising" is to be construed as inclusive and open-ended and not as exclusive. In particular, when used in this specification including the claims, the term "comprising" and variations thereof mean that the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

The foregoing detailed description of the preferred embodiments and advantages of the invention will be appreciated that the foregoing description is merely illustrative of the presently preferred embodiments of the invention, and that no changes, additions, substitutions and equivalents of those embodiments are intended to be included within the scope of the invention.

Claims

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

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: