Coherent raman microscopic imaging method based on planar parallel microcavity

Abstract

The present application belongs to the field of optical microscopic imaging technology, and particularly relates to a coherent Raman microscopic imaging method based on a plane-parallel microcavity. The present application first applies a plane-parallel microcavity structure, i.e. a Fabry-Perot resonant cavity, to a traditional coherent Raman microscopic imaging, designs a microcavity structure with different reflectivity coating, and respectively controls coherent anti-Stokes Raman scattering and stimulated Raman scattering processes, so as to couple spatial structure information of a sample and Raman signals to generate new spectra and images, and respectively realize cavity-enhanced coherent anti-Stokes Raman scattering imaging and cavity-enhanced stimulated Raman scattering imaging. Compared with the traditional coherent Raman microscopic imaging, the present application improves signal intensity, and simultaneously acquires spatial structure information and chemical information of a sample, so as to provide more dimensional information of the sample, and compared with the traditional biological microcavity sensing imaging, can realize fast and label-free imaging, and has wide application prospects in the fields of biomedical detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of optical microscopy imaging technology, specifically relating to a coherent Raman microscopy imaging method based on a planar parallel microcavity. Background Technology

[0002] Coherent Raman scattering (CRS) imaging is a common nonlinear optical microscopy technique that enhances Raman signals through the nonlinear coherent Raman effect, enabling high-resolution, high-sensitivity molecular imaging. It primarily includes stimulated Raman scattering (SRS) imaging and coherent anti-Stokes Raman scattering (CARS) imaging. Due to their label-free and non-invasive nature, these two techniques are widely used in biomedicine, chemical analysis, and materials science. Although both utilize the characteristics of coherent Raman scattering, they differ in signal generation mechanisms, imaging characteristics, and application scenarios. Compared to traditional optical microscopy, CRS imaging can provide rich information on the chemical composition of samples, helping scientists understand changes in the composition of various substances during biological processes. Compared to staining and fluorescent labeling methods, CRS imaging avoids the effects of external fluorescent labels on biological cells and the problems of photobleaching damage, making it a safer and more efficient approach.

[0003] An optical cavity is a device used to store and amplify light waves, widely used in lasers, fiber optic communications, and sensing technologies. It consists of two or more mirrors, and the light waves reflected back and forth between the mirrors form standing wave modes. Optical cavities can adjust and control the frequency, phase, and intensity of the light field, providing many important functions, such as increasing light-matter interaction, achieving high-precision measurements, and generating lasers. The working principle of an optical cavity is based on the phenomenon of interference. When a light wave enters the cavity, it reflects multiple times between the mirrors, forming a stable interference mode, also known as a resonant mode. These resonant modes correspond to specific frequencies, depending on the cavity's geometry and material properties. When the frequency of the input light matches the cavity's resonant frequency, the light wave is stored and circulates within the cavity, forming a high-intensity electromagnetic field. The Fabry-Pérot cavity is a classic optical resonator consisting of two parallel plane mirrors, also known as a parallel plane cavity. It has advantages such as simple structure and easy fabrication, but it has high diffraction loss and requires high alignment accuracy. Therefore, it is often used in cavities with short spacing, such as microchips and microcavity lasers. Its planar emission characteristics also make it commonly used in optical sensing, especially imaging.

[0004] In recent years, embedding biomaterials within gain media and optical cavities has emerged as a novel technique offering new methods for utilizing optical cavities in biological and biomedical applications. Because optical cavities provide strong light-matter interactions, subtle variations in the bio-gain medium can be amplified, resulting in unique output laser characteristics such as narrow linewidth, high intensity, and high contrast. Recent advances in cellular lasers have sparked widespread interest in cell tracking, cell type analysis, intracellular detection, and phenotyping. In particular, single-cell lasers utilizing planar parallel microcavities offer the advantage of whole-body interaction between the electromagnetic field and the gain medium, providing a sensitive method for studying the physical structure and interactions of cells.

[0005] Traditional biolasers use external fluorescent labels as gain media to achieve interaction with the optical cavity. However, external fluorescent labels can affect biological samples and cause photobleaching during imaging. Furthermore, external labels complicate the sample preparation process.

[0006] Traditional coherent Raman scattering microscopy, whose signal originates from the vibration of specific chemical bonds, allows us to rapidly extract the spatial chemical information of samples without labels. While chemical information is important in biology, spatial structural information is also crucial. However, this technique cannot effectively acquire structural information. Furthermore, label-free coherent Raman scattering imaging suffers from problems such as low contrast and low signal-to-noise ratio.

[0007] In recent years, optical microcavities have been applied to the biomedical field. The cavity interacts with matter, generating resonance at specific frequencies, which enhances the resonance signal. According to the resonance conditions, this is related to the material's structural properties; therefore, the emitted light carries information about the material's spatial structure. Combined with coherent Raman microscopy, this eliminates the need for external fluorescent markers as gain media. This invention, through cavity structure design, achieves selective cavity enhancement and conversion of both CARS and SRS channels. Summary of the Invention

[0008] The purpose of this invention is to provide a coherent Raman scattering microscopy imaging method based on a planar parallel microcavity, so as to solve the problems of weak signals in coherent Raman scattering microscopy imaging and the inability to obtain structural information by relying solely on chemical information imaging.

[0009] The coherent Raman scattering microscopy imaging method based on parallel microcavities provided by this invention combines planar parallel microcavities with coherent Raman scattering microscopy imaging technology, and selectively establishes cavity enhancement and conversion for both CARS and SRS channels: the microcavity is coupled to the coherent anti-Stokes Raman scattering process (FP-CARS) without affecting the stimulated Raman scattering process; the microcavity is coupled to the stimulated Raman scattering process (FP-SRS); the specific steps are as follows:

[0010] S1. Planar parallel microcavities (hereinafter referred to as microcavities) with different reflectivities are selected to couple coherent anti-Stokes Raman scattering processes or stimulated Raman scattering processes, depending on the experimental requirements.

[0011] S2. Based on the microcavity selected in step S1, set the image acquisition mode, such as... Figure 1 As shown in a: If the microcavity is coupled with the coherent anti-Stokes Raman scattering process, the coherent anti-Stokes Raman scattering signal is detected from the back and the stimulated Raman scattering signal is detected from the front; if the microcavity is coupled with the stimulated Raman scattering process, the stimulated Raman scattering signal is detected from the front.

[0012] S3. Place the sample to be tested into the microcavity and fix the cavity length using polystyrene microspheres;

[0013] S4. Place the microcavity into the stage, select pump light and Stokes light with corresponding frequency differences according to the chemical composition of the analyte, so that it generates coherent Raman signals corresponding to different Raman frequencies, and the detector acquires the signals to form an image.

[0014] The planar parallel microcavity described in this invention is also known as a Fabry-Perot cavity [1], such as Figure 1 As shown in b, the microcavity consists of two plane mirrors with coatings that have specific reflectivities for different light bands and can be selectively coupled to two different processes: coherent anti-Stokes Raman scattering or stimulated Raman scattering. If coupled with coherent anti-Stokes Raman scattering, it is designed to have high reflectivity (e.g., above 99%) in the coherent anti-Stokes Raman scattering band and high transmittance (e.g., above 95%) in the pump and Stokes bands. This confines the coherent anti-Stokes light within the cavity to resonate, thereby generating an FP-CARS signal. This does not affect the pump and Stokes light, and therefore does not affect the stimulated Raman scattering process. If coupled with stimulated Raman scattering, the pump and Stokes light are designed to have high reflectivity (e.g., currently 70% reflectivity for the pump light and 90% reflectivity for the Stokes light are commonly used). This causes the pump and Stokes light to oscillate within the cavity, generating an FP-SRS signal. Since it serves as both the pump light and the signal light to be detected, the reflectivity cannot be designed to be too high. Furthermore, this invention uses demodulation of the pump light to obtain the signal, so the reflectivity of the pump light is designed to be lower than that of the Stokes light.

[0015] Polystyrene microspheres are placed around the perimeter of the microcavity to fix its length. Specifically, the cavity length (i.e., the distance between the two plane mirrors) can be varied by using polystyrene microspheres of different diameters. Generally, 10µm or 15µm diameter microspheres are commonly used to fix the cavity length. Since typical cell or tissue samples are in the 5-10µm range, the cavity length is slightly larger than the sample size and is fixed to improve the parallelism and stability of the cavity. The sample to be tested is placed inside the cavity, and the microcavity is placed on the stage for imaging. The stage, located between the objective lens and the condenser lens, is used to hold the sample.

[0016] This invention combines planar parallel microcavities with coherent Raman scattering microscopy and selectively establishes cavity enhancement and conversion for both CARS and SRS channels; 1. Coupling the microcavity with the coherent anti-Stokes Raman scattering process (FP-CARS) without affecting the stimulated Raman scattering process; 2. Coupling the microcavity with the stimulated Raman scattering process (FP-SRS).

[0017] The present invention has the following advantages:

[0018] (1) The image data shows the transverse resonance modes of various coherent Raman scattered light from polystyrene microspheres, achieving a 14-fold improvement in the emitted FP-CARS signal compared to the back-facing CARS.

[0019] (2) Due to the control of the microcavity, the Raman spectral lines are coupled with the spatial structure information of the cells, enabling multimodal detection of cell samples. Simultaneously, CARS signal reflection reception and SRS signal transmission reception are performed. Due to the differences in subcellular structures, the optical cavity improves the optical contrast between different structures, such as the cell nucleus and cytoplasm. It can simultaneously acquire the subcellular three-dimensional spatial structure information and multidimensional chemical information of biological samples, solving the problem that the original coherent Raman scattering imaging could only acquire the chemical distribution of biological samples and could not intuitively display the structural information of the samples.

[0020] (3) For the first time, the observation of FP-SRS imaging was realized. The coupling of spatial structure information and chemical information produced new spectra and images. The more stringent resonance conditions made the contrast changes brought about by the structure information more obvious.

[0021] Compared to existing coherent Raman scattering microscopy, this invention combines a planar parallel microcavity, enhancing signal intensity and enabling the simultaneous acquisition of spatial and chemical information of the sample. This provides more dimensional information about the sample. Compared to traditional biological microcavity sensing imaging, combining the cavity with the coherent Raman scattering process eliminates the need for external markers, allowing for rapid, label-free imaging. The enhanced signal and acquisition of more dimensional information make planar parallel microcavity-based coherent Raman scattering microscopy a promising candidate for applications in biomedical detection and other fields. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the microcavity structure and physical processes.

[0023] Figure 2 This image shows a microcavity-based coherent Raman microscopy system and its imaging results.

[0024] Figure 3 An example of FP-CARS imaging of polystyrene microspheres and related results.

[0025] Figure 4 Example of FP-CARS imaging of cervical cells and related results.

[0026] Figure 5 Examples of FP-SRS imaging and related results are provided. Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0028] This invention provides a coherent Raman scattering microscopy imaging method based on a planar parallel microcavity. First, a system is constructed as follows... Figure 2 The coherent Raman scattering microscopy system shown [2]. Pump light and Stokes light are incident as excitation sources. Stokes light is modulated to 20MHz by an electro-optic modulator, and pump light is kept constant at 80MHz. It is incident into the microscope and scanned by a scanning galvanometer. Coherent Raman scattering occurs between the sample on the stage behind the objective lens. The coherent anti-Stokes Raman scattering signal is received by a photomultiplier tube by back-probe. The filter is a bandpass filter suitable for CARS signals. The stimulated Raman scattering signal is detected by a photodetector after the Stokes light is filtered out by the filter by forward-probe. Then, the stimulated Raman scattering signal is demodulated from the pump light by a lock-in amplifier to obtain the stimulated Raman scattering signal.

[0029] In this invention, the microcavity can selectively act on either coherent anti-Stokes Raman scattering or stimulated Raman scattering. When the microcavity is selectively acted on coherent anti-Stokes Raman scattering, the cavity coating is designed to have high reflectivity (99.3%) for the coherent anti-Stokes Raman scattering light band and high transmittance (greater than 95%) for the pump and Stokes light bands. When the microcavity is selectively acted on stimulated Raman scattering, the cavity coating is designed to have high reflectivity for both the pump and Stokes light (typically 70% reflectivity for the pump light and 90% for the Stokes light); polystyrene microspheres are used to fix the cavity length.

[0030] The specific steps are as follows:

[0031] S1. Place a coated plane mirror on a glass slide, and drop polystyrene microspheres of appropriate concentration into the four corners of the plane mirror to ensure that the microsphere density is appropriate and that they do not overlap.

[0032] S2. Place the sample to be tested in the center of the plane mirror. Here, polystyrene microspheres and cervical cells are used as the sample to be tested. Place another coated plane mirror on top of it, then place a coverslip on top and gently press to make the solution in the cavity uniform.

[0033] S3. Imaging is performed by changing the wavenumber difference between the pump light and the Stokes light to correspond to different Raman signals.

[0034] This invention demonstrates the feasibility of combining planar parallel microcavities with coherent Raman scattering microscopy through illustrative analysis of data on polystyrene microspheres and cervical cells within microcavities.

[0035] For FP-CARS, such as Figure 2 When the cavity is coupled to coherent anti-Stokes Raman scattering without affecting stimulated Raman scattering, a transverse resonance mode is generated due to the coupling between the microcavity and the coherent anti-Stokes light, while the stimulated Raman scattering image in the same field of view remains unchanged.

[0036] Figure 3 middle, Figure 3 a demonstrates the various transverse resonance modes generated after the cavity is coupled with CARS. Figure 3 b shows the microsphere images with and without cavities, and compares the Raman signals of the microspheres with and without cavities. The results show that the signal is enhanced by 14 times under the influence of the microcavity. Figure 3 c shows the new spectral lines that couple structural information between the cavity and CARS. It can be seen that microspheres of different sizes exhibit different new spectral lines, indicating that the new spectral lines reflect the structural information of the microspheres.

[0037] Figure 4 In this study, cervical cells were used as the test sample. Due to their structural differences, the nucleus and cytoplasm of these cells were separated through interaction with microcavities. Figure 4 a represents the SRS and FP-CARS spectra of the cell nucleus and cytoplasm. It can be seen that there is no significant difference between the SRS spectra of the cell nucleus and cytoplasm, indicating that their chemical composition is not significantly different. However, the FP-CARS spectra show a significant frequency shift, which is due to the different spatial structures of the cell nucleus and cytoplasm, resulting in different spectral lines when coupled with the cavity. Figure 4 b and c show FP-CARS images at the new spectral peak positions, with the cell nucleus and cytoplasm enhanced respectively. Figure 4 d is the SRS image of the cell. Since there is a stimulated Raman scattering image in the same field of view, and the cavity designed for CARS does not affect the SRS process, its chemical and structural information can be obtained simultaneously.

[0038] Figure 5The data on microspheres and cells from FP-SRS were presented, resulting in new images and spectra. Figure 5 Image a demonstrates FP-SRS imaging of polystyrene microspheres, revealing numerous lateral electromagnetic modes, indicating coupling between the cavity and the SRS process. Figure 5 b shows the FP-SRS image of cervical cells as the test sample. Similar to the results of FP-CARS, due to the structural differences between the cell nucleus and cytoplasm, images with enhancement of the cell nucleus and cytoplasm were generated respectively. Furthermore, since FP-SRS modulates both pump light and Stokes light, the resonance conditions are more stringent. Compared to FP-CARS images, the cytoplasmic enhancement in FP-SRS images is accompanied by stronger suppression of the cell nucleus. Figure 5 c shows the SRS and FP-SRS spectra of the cell nucleus and cytoplasm. Similar to FP-CARS, due to the different structures of the cell nucleus and cytoplasm, this information is coupled into the SRS spectra, thus producing different spectral frequency shifts.

[0039] Based on the above embodiments, the coherent Raman scattering microscopy imaging method based on planar parallel microcavities can be summarized as follows: constructing a coherent Raman scattering microscopy imaging optical path; placing the sample to be tested inside a specially designed microcavity; and acquiring images.

[0040] The examples selected only polystyrene microspheres and cervical cells as sample analytes to illustrate the experimental concept and characteristics of the present invention. The scope of protection of the present invention is not limited to the above examples. All equivalent changes or modifications made based on the principles and experimental ideas disclosed in this invention are within the scope of protection of this invention.

[0041] References

[0042] 1. Wu,

[0043] 2. Liu, Z. et al. Instant diagnosis of gastroscopic biopsy via deep-learned single-shot femtosecond stimulated Raman histology. Nature Communications 13, 4050 (2022). https: / / doi.org:10.1038 / s41467-022-31339-8 .

Claims

1. A coherent Raman scattering microscopy imaging method based on a planar parallel microcavity, characterized in that, The planar parallel microcavity is combined with coherent Raman scattering microscopy, and cavity enhancement and conversion of the CARS / SRS two channels are selectively established: the microcavity is coupled to the coherent anti-Stokes Raman scattering process without affecting the stimulated Raman scattering process; or, the microcavity is coupled to the stimulated Raman scattering process; the specific steps are as follows: S1. Planar parallel microcavities with different reflectivities are selected to couple coherent anti-Stokes Raman scattering or stimulated Raman scattering processes, depending on the experimental requirements. S2. Based on the microcavity selected in step S1, set the image acquisition mode: if the microcavity is coupled with the coherent anti-Stokes Raman scattering process, then the coherent anti-Stokes Raman scattering signal is detected from the back and the stimulated Raman scattering signal is detected from the front; if the microcavity is coupled with the stimulated Raman scattering process, then the stimulated Raman scattering signal is detected from the front. S3. Place the sample to be tested into the microcavity and fix the cavity length using polystyrene microspheres; S4. Place the microcavity into the stage, select pump light and Stokes light with corresponding frequency differences according to the chemical composition of the analyte, so that it generates coherent Raman signals corresponding to different Raman frequencies, and the detector acquires the signals to form an image. The aforementioned planar parallel microcavity consists of two plane mirrors coated with specific reflectivities for different light bands. These mirrors are controlled for two different processes: selective coupling with either coherent anti-Stokes Raman scattering (ARS) or stimulated Raman scattering (SRS). For ARS coupling, the mirror is designed to have high reflectivity in the ARS band and high transmittance in the pump and Stokes bands, confining the ARS beam within the cavity to resonate and generate an FP-CARS signal without affecting the pump and Stokes beams. For SRS coupling, the mirror is designed to have high reflectivity in both the pump and Stokes beams, causing them to oscillate within the cavity and generate an FP-SRS signal.

2. The coherent Raman scattering microscopy imaging method based on planar parallel microcavities according to claim 1, characterized in that, The method of using polystyrene microspheres to fix the cavity length specifically involves using polystyrene microspheres of different diameters to change the cavity length, i.e., the distance between the two plane mirrors, so that the cavity length is slightly larger than the sample size and is then fixed.

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