Single bacterial detection method based on raman spectroscopy technology through dmd and microfluidics

Abstract

The present application relates to a kind of single bacterial detection methods based on Raman spectroscopy technique through DMD and microfluidic, laser, DMD, microscope, CCD camera, microfluidic chip and Raman spectrum detection analysis platform are used, the method injects the microorganism sample to be measured into microfluidic chip, captures cell by DMD-based optical tweezers technology, collects the Raman spectrum of microorganism cell, processes spectral data, judges whether microorganism exists according to spectral characteristics and its kind, metabolic state information.The present application has the characteristics of non-invasive, high sensitivity, can realize rapid high-throughput single bacterial in situ detection, and has important significance for bacterial detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of spectral detection, and particularly relates to a single bacterial detection method based on Raman spectroscopy technology through a DMD and microfluidics. BACKGROUND

[0002] Currently, the importance of bacterial detection spans across the fields of medicine, environment, food safety, etc. It helps to diagnose diseases, protect public health, ensure food and water safety, and monitor environmental pollution. Traditional bacterial detection methods mainly rely on culture, microscopic observation, biochemical tests, etc. These methods are time-consuming, complex to operate, low in sensitivity, and susceptible to interference, etc. In recent years, some new microbial detection methods have been developed, such as molecular biology and immunology methods based on PCR, ELISA, mass spectrometry, etc. These methods have the advantages of high sensitivity, high specificity, high throughput, etc., but also have some limitations. These methods require tedious pretreatment and detection operations, require bacterial culture or staining, are time-consuming and high in cost, and molecular biology detection methods also need to destroy the bacterial structure, making it difficult to be used for low-abundance microbial detection in situ.

[0003] In recent years, Raman spectroscopy technology has been widely applied in the field of bacterial detection. Raman spectroscopy is a scattering spectrum caused by the rotation and vibration modes of molecular bonds. Through spectral analysis, information about the molecular structure can be obtained. It does not require sample preparation, and any gaseous, liquid, or solid sample can be directly measured through a fiber probe or through glass, quartz, and optical fiber. It can provide rapid, simple, repeatable, and more importantly, non-destructive qualitative and quantitative analysis, and is an important means for analyzing the structure of organic compounds. Raman spectroscopy can effectively identify the biochemical components of microorganisms, thereby obtaining the "whole cell fingerprint" of bacteria. This technology has a wide range of applications in microbial detection and analysis, and is particularly suitable for rapid diagnosis of the cause and "precision medicine". Specifically, Raman spectroscopy can provide information about the biological macromolecular components of bacteria, such as nucleic acids, proteins, and lipids, forming phenotypic characteristics at the single-cell level. It can distinguish the molecular composition of different microorganisms, such as the composition of protein groups, lipids, carbohydrates, or cell walls, thereby achieving the identification and classification of microorganisms. As a rapid, non-destructive, and non-contact technology, Raman spectroscopy has important significance for microbial detection and research, and is expected to play a greater role in the fields of clinical diagnosis, food safety, and environmental monitoring.

[0004] However, traditional Raman spectroscopy techniques usually require multiple detectors to measure Raman scattering signals to improve signal-to-noise ratio and resolution, which increases system cost and complexity. At the same time, it is usually a collective detection of a large number of bacteria, which cannot realize single-cell level detection, and the signal intensity is usually low, which requires a long integration time to obtain sufficient signal, which is not ideal for rapid detection of single bacteria. The background signal of the sample (such as fluorescence, scattering, etc.) may interfere with the Raman signal, reducing the accuracy of the detection. On the other hand, cells in a liquid will undergo Brownian motion, which is a random motion caused by molecular collisions. Therefore, when using the commonly used optical tweezer technology to clamp the cells, enough force needs to be applied to resist the Brownian motion to ensure that the cells are stably captured. And the laser beam has a certain energy on the biological tissue, which may cause photo damage to the cells when using the optical tweezer technology. Photo damage may include thermal effects, photochemical effects, or other irreversible effects.

[0005] Globally, pathogenic microorganisms such as fungi, bacteria and viruses have been one of the important causes of death in developed and developing countries, and infectious diseases caused by pathogenic bacteria are still one of the most important global public health problems. Rapid and accurate identification of pathogenic bacteria and their drug resistance is crucial for disease diagnosis, treatment effect evaluation, drug dosage guidance and infectious disease prevention and control. Therefore, developing new and efficient single-bacterium analysis methods is crucial for public health.

[0006] To solve this problem, the present application provides a single bacterium detection method based on Raman spectroscopy technology through DMD and microfluidics. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art, provide a single bacterium detection method based on Raman spectroscopy technology through DMD and microfluidics, aiming at the rapid high-throughput detection demand of microorganisms, using single bacterium Raman spectroscopy imaging technology, developing high-throughput preliminary screening technology based on laser diffraction and microfluidics, DMD-based optical tweezer technology, realizing functional imaging of single bacterium, mining the component structure details of different bacteria, solving the problem of interference of complex components in the sample to be detected, improving the detection specificity of the technology, and realizing non-invasive, high-sensitivity, rapid and non-destructive high-throughput detection of single bacterium.

[0008] The technical problem of the present application is solved by the following technical scheme:

[0009] A single bacterium detection method based on Raman spectroscopy technology through DMD and microfluidics, which adopts a laser, a DMD, a microscope, a CCD camera, a microfluidic chip and a Raman spectroscopy detection and analysis platform, and the steps of the method are:

[0010] S1, collect the microorganism sample to be tested, prepare the sample bacterial liquid, so as to be processed in the microfluidic chip;

[0011] S2, select a microfluidic chip with appropriate channel depth and width, inject the prepared bacterial liquid into the microfluidic channel, and construct a stable static flow field with a flow rate of zero in the microfluidic chip, and limit the sample cells;

[0012] S3, select a laser with an appropriate wave band as a laser light source, excite a Raman spectrum signal, and the light path enters the microfluidic chip injected with the bacterial liquid to be tested;

[0013] S4, after the light path is expanded, the reflection and transmission of the light path are realized by the optical filter to realize different functions, and the Kohler illumination is adopted to provide uniform and bright illumination;

[0014] S5, the imaging pattern and its characteristics are regulated by rotating the micro-mirror of the DMD around the fixed shaft, the light beam is focused on the cell to be tested, the optical tweezers function is realized, the cell to be tested is captured, and then the Stokes scattering signal reflected by the cell is collected, that is, the Raman spectrum of the microorganism cell;

[0015] S6, the collected Raman spectrum is subjected to noise removal and background correction processing, and the type and characteristics of the microorganism are determined by comparing the known microorganism spectrum database;

[0016] S7, according to the spectral characteristics, whether the microorganism exists and the type, metabolic state information of the microorganism are judged.

[0017] Moreover, the microfluidic chip is a polydimethylsiloxane (PDMS) microfluidic chip, the chip polydimethylsiloxane (PDMS) microfluidic chip has different channel widths of 50 μm, 100 μm, 200 μm and a uniform channel depth of 50 μm.

[0018] Moreover, the resolution of the micro-mirror of the DMD is 1280*800.

[0019] Moreover, the wavelength of the laser light source is 633 nm.

[0020] Moreover, the optical filter is a specific narrow band pass filter and a long wave pass filter.

[0021] Moreover, the S4 uses 600 nm band rejection, 633 nm long wave pass filter and 490 nm long wave pass filter to separate the reflected light path, one of which is used for microscope imaging, and the other is used for collecting the Raman spectrum signal.

[0022] The advantages and beneficial effects of the present application are:

[0023] 1、The application selects polydimethylsiloxane (PDMS) microfluidic chip. PDMS material is widely used in the construction of microfluidic system due to its excellent biocompatibility and transparency. In the microfluidic chip, a stable static flow field with zero flow rate is constructed to confine the sample cells. The establishment of static flow field enables the sample cells to be effectively confined in the chip, thereby avoiding the change of cell position caused by flow, ensuring the accuracy of spectral data. In addition, the establishment of static flow field also provides a stable environment for subsequent Raman spectrum analysis, so that we can more accurately capture the spectral characteristics of cells.

[0024] 2、The application develops a light tweezer technology based on DMD. By controlling the rotation of the tiny mirror of DMD around the fixed axis, the imaging pattern and its characteristics are adjusted, the light beam is focused on the cell to be measured, and the light tweezer function is realized to capture the cell to be measured. Then the Stokes scattering signal reflected by the cell is collected, that is, the Raman spectrum of the microbial cell. Using this method, the rotation of the DMD micromirror unit can be controlled by the host computer to adjust the spot shape according to the shape and other characteristics of the cell to be measured, so that the laser energy can simultaneously meet the requirements of the light tweezer to provide appropriate size of force to resist the Brownian motion of the cell to ensure that the cell is stably captured, and the laser energy can be avoided to be too strong to cause photo damage to the cell, so as to improve the stability, accuracy and repeatability of detection.

[0025] 3、The application uses 633nm laser as the excitation light source for Raman spectrum analysis. This choice is based on the advantages of 633nm laser, including its ability to provide higher scattering intensity, thereby enhancing the detection sensitivity of the signal; smaller spot diameter helps to improve the spatial resolution; and suitable for suppressing the fluorescence interference in biological samples. In addition, the high stability and excellent beam quality of 633nm laser system are also crucial to ensure the accuracy and repeatability of Raman spectrum analysis. These characteristics make 633nm laser an ideal light source for Raman spectrum analysis in the field of biomedicine, especially in situations requiring high sensitivity and high spatial resolution. Through this method, we can more accurately capture the spectral characteristics of cells, providing a reliable data basis for biological molecular structure analysis and biomedical research. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The flowchart of the application;

[0027] Figure 2 The result schematic diagram of the application. DETAILED DESCRIPTION

[0028] The application will be further described in detail below through specific embodiments, the following embodiments are only descriptive and not limiting, and cannot limit the protection scope of the application.

[0029] The present application proposes a single bacterial detection method based on Raman spectroscopy technology through DMD and microfluidics. First, collect the microbial sample to be tested, prepare the sample liquid for processing in the microfluidic chip. Select a microfluidic chip with appropriate channel depth and width, inject the prepared bacterial liquid into the microfluidic channel, ensure its structure and function are normal, and construct a stable static flow field with zero flow rate in the microfluidic chip to confine the sample cells. Select 633nm laser as the laser light source to excite Raman spectrum signal, and the light path enters the microfluidic chip injected with the bacteria to be tested. After expansion, the light path passes through the optical filter to realize different functions of reflection and transmission, and adopts Kohler illumination technology to provide uniform and bright illumination. By controlling the rotation of the micro-mirror of the DMD around the fixed axis to regulate the imaging pattern and its characteristics, the light beam is focused on the cell to be tested to realize the function of optical tweezers and capture the cell to be tested. Further collect the Stokes scattering signal reflected by the cell, i.e. the Raman spectrum of the microbial cell; the collected Raman spectrum signal is subjected to a series of preprocessing steps, including noise removal, background correction, signal normalization and application of smoothing algorithm, to improve the signal-to-noise ratio and resolution of the signal. The processed spectrum data is compared with the known microbial spectrum database to determine the type and characteristics of the microorganism. This method can not only quickly determine the presence or absence of microorganisms, but also provide detailed information about the type, metabolic state, etc. of the microorganism. The test sample can simultaneously output different bacterial detection indicators in a short time through signal excitation, Raman spectrum acquisition, spectrum reconstruction, single bacterial Raman spectrum imaging and bacterial concentration analysis. According to the experimental data, the total number of colonies, coliform group, Staphylococcus aureus, Salmonella and other specified detection indicators can be detected simultaneously in only a few hours.

[0030] A single bacterial detection method based on Raman spectroscopy technology through DMD and microfluidics, the innovation of the method steps is:

[0031] 1、Prepare a proper concentration of E. coli bacteria solution, and add phosphate buffered saline solution (PBS) to adjust the concentration to 10^6 cfu / ml. This concentration is considered ideal for Raman spectroscopy analysis as it provides sufficient number of cells without causing excessive aggregation, which can interfere with the spectral signal. Three different channel widths (50 μm, 100 μm, and 200 μm) and a uniform channel depth (50 μm) of polydimethylsiloxane (PDMS) microfluidic chips are selected. PDMS is widely used in the construction of microfluidic systems due to its excellent biocompatibility and transparency. An injection pump is used to inject the prepared bacteria solution into the microfluidic channel. The maximum pressure during injection is strictly controlled not to exceed 2 bar, and the maximum flow rate is not to exceed 2 ml / min to prevent leakage of the chip and ensure the smooth progress of the experiment. A stable static flow field with a flow rate of zero is established in the microfluidic chip to confine the sample cells. The establishment of a static flow field allows the sample cells to be effectively confined in the chip, avoiding changes in cell position caused by flow, ensuring the accuracy of the spectral data. In addition, the establishment of a static flow field also provides a stable environment for subsequent Raman spectroscopy analysis, allowing us to more accurately capture the spectral characteristics of the cells.

[0032] 2、Select 633 nm laser as the laser source, which is based on the advantages of 633 nm laser. The Raman scattering intensity is inversely proportional to the fourth power of the laser wavelength, so 633 nm visible light laser can provide stronger scattering intensity than near-infrared laser (such as 785 nm or 1064 nm), thereby enhancing the detection sensitivity of the signal; under the diffraction limit condition, the diameter of the laser spot depends on the wavelength of the excitation laser and the numerical aperture of the microscope objective. 633 nm laser can provide a smaller spot diameter, thereby improving the spatial resolution to some extent; in addition, the single-mode laser emission provided by the 633 nm laser system has a high coherence length and a high side-mode suppression ratio, and the wavelength stability is better than 0.015 nm. The high stability and excellent beam quality of 633 nm laser are crucial for ensuring the accuracy and repeatability of Raman spectroscopy analysis, and are suitable for suppressing fluorescence interference in biological samples. These characteristics make 633 nm laser an ideal light source for Raman spectroscopy analysis in the field of biomedicine, especially in situations requiring high sensitivity and high spatial resolution. Through this method, we can more accurately capture the spectral characteristics of the cells, providing a reliable data basis for the analysis of biological molecular structure and biomedical research.

[0033] The light path enters the microfluidic chip where the bacteria solution is injected, and the bacteria produce Raman scattering effects on the input signal, thereby changing the frequency and intensity of the input signal. As shown in Figure 2The 633 nm laser is emitted from a laser, and a lens is used to achieve beam expansion, which can increase the diameter of the laser beam and reduce the power density of the beam, thereby reducing the risk of laser-induced damage to the sample or optical elements. This beam expansion also improves the uniformity of the beam and improves the overall beam quality. In addition, the expanded beam is easier to focus into a smaller spot, which is crucial for improving the spatial resolution of the Raman signal. The expanded beam is also more suitable for matching with optical elements in the subsequent optical path, such as filters and gratings, to maximize the performance of the optical system. When designing a Raman optical system, parameters such as the focal length of the lens, the beam diameter, and the beam divergence angle need to be considered comprehensively to ensure that the beam expansion effect meets the system requirements.

[0034] 3、The expanded beam first passes through a specific narrow-bandpass filter. This filter has a center wavelength of 632.8 nm and is designed to work at an incident angle of 0±2° to ensure that the 633 nm red light is transmitted while effectively blocking the 582-623 nm and 642-696 nm wavelength bands. This selective transmission mechanism is crucial for improving the spectral selectivity and signal contrast of the system. Subsequently, the beam in the optical path further passes through a 490 nm long-wave pass filter, which works at an incident angle of 45° and allows the transmission of 505-800 nm wavelength band light while reflecting 380-475 nm wavelength band light. Such a design not only ensures that the 633 nm laser light can pass smoothly, but also reflects the reflected light from the cells to be measured and the monochromatic light used for illumination, thereby providing sufficient illumination conditions for the cells to be measured and allowing clear imaging in the CCD camera.

[0035] In addition, the system uses Kohler illumination technology, which can provide uniform and bright illumination for the measured area while avoiding possible glare problems. The application of a beam splitter further optimizes the optical path, which allows the transmission of monochromatic illumination light to illuminate the measured area while guiding the reflected light from the measured area to the CCD camera, achieving accurate observation and imaging of cell features. This comprehensive optical path design significantly improves the performance of the Raman optical system, making it more accurate and efficient in biomedical imaging and spectral analysis. The application of these technologies not only optimizes the optical path but also provides a solid foundation for high-quality optical imaging.

[0036] 4. By controlling the rotation of the tiny reflecting mirrors of the 1280x800 resolution DMD around a fixed axis, the imaging pattern and its characteristics are adjusted, focusing the light beam onto the cell to be tested, thus achieving the function of optical tweezers to fix and capture the cell. During this process, the Stokes scattering signal reflected by the cell is effectively received, which is the Raman spectrum of the measured microbial cell. By using a 633nm long-pass filter and a 600nm band-stop filter, we can split the reflected light path into two paths. One path is reflected back to the CCD camera, presenting a microscope image in the corresponding camera software on the computer for easy observation; the other path passes through the 633nm long-pass filter into the Raman spectrometer to collect the Raman spectral signal and perform spectral analysis.

[0037] A Discrete Micromirror Device (DMD) is primarily an array of micromirror units, each with its own independent structure. Its resolution depends on the number of micromirror units. Each micromirror unit in a DMD consists of a data storage unit, a rotating axis, a reflective mirror, and other connecting structures. The micromirrors are fixed to a flip-up platform, allowing the flipping of the micromirror units to be controlled by a drive control circuit, thus determining the path of the reflected light. Furthermore, the micromirror unit structure is integrated into a CMOS address circuit, facilitating the supply of drive voltages and control commands to the DMD.

[0038] Wave optics theory is the fundamental theory of optical tweezers. It posits that two forces exist along the optical axis: (1) a scattering force, which is parallel to the incident light and proportional to the light intensity; and (2) a gradient force, parallel to the light field gradient and proportional to the intensity gradient. A Rayleigh particle with refractive index m is placed in a medium with refractive index n1. The scattering force Fs that pushes it away from the focal point is expressed as:

[0039] F s =n1P s / c

[0040] In the formula, Ps represents the light power scattered by the particle. When a spherical Rayleigh particle is located in the light field, it is subjected to a gradient force Fg pointing towards the focal point, which is expressed as:

[0041]

[0042] Where α represents molecular polarizability.

[0043] To form a potential well capable of trapping the Rayleigh particle, the gradient force Fg (pulling the particle toward the focal point) must be greater than the scattering force Fs (pushing the particle away from the focal point), i.e., the following formula must be satisfied:

[0044]

[0045] The host computer controls the rotation of the DMD micromirror unit to adjust the spot shape according to the shape of the cell to be detected, so that the laser energy can meet the requirements of providing appropriate size of force as optical tweezers to resist the Brownian motion of the cell to ensure that the cell is stably captured, and the laser energy can be avoided to be too strong to cause optical damage to the cell, so as to improve the stability, accuracy and repeatability of detection.

[0046] The same sample is irradiated by light of different frequencies, and the change amount Δν of the Raman scattered light relative to the frequency of the incident light remains unchanged, which is called the Raman shift and is a direct manifestation of the molecular vibration information. The calculation formula of the Raman shift can be expressed as:

[0047] Δv=v0-v R

[0048] Where Δν is the Raman shift, ν0 is the frequency of the incident light, and νR is the frequency of the Raman scattered light. The Raman shift is only related to the molecular vibration group of the substance, and is independent of the frequency of the incident light. Therefore, the Raman shift is very suitable for qualitative analysis of substances, and the Raman spectrum technology is a substance analysis method based on Raman scattering and Raman shift. Spectral analysis technology plays an important role in many fields such as chemistry, life science and food safety. When studying clinical pathogenic bacteria, the application of Raman spectrum technology has the following advantages:

[0049] (1) Not affected by water. Since pathogenic bacteria samples are usually present in liquid, the strong infrared absorption of water will affect the infrared spectrum analysis of other substances in pathogenic bacteria. Therefore, pathogenic bacteria samples in liquid are not suitable for infrared spectrum analysis. Raman spectrum method is an ideal tool for studying aqueous materials, because the asymmetry of water molecule chemical bond can avoid the influence of too strong infrared absorption to make the Raman signal intensity very weak. Therefore, Raman spectrum method is very suitable for studying water system, and is an ideal technology for studying biological samples and chemical compounds in aqueous solution.

[0050] (2) Non-destructive to sample material. By properly adjusting the laser power, Raman collection time and other parameters, Raman spectrum method can be used for non-destructive research of bacteria. In order to obtain high-quality bacterial Raman signal and avoid damage to the chemical structure of bacteria, low laser energy and short detection time should be used in Raman experiment, and gradually increased until high signal-to-noise ratio Raman signal is obtained without damaging the sample.

[0051] (3) Realize single cell level detection. The magnification of Raman microscope can reach 100 times, the spot diameter of laser can be less than 1 pm, and the resolution can reach 0.4 pm. The diameter of most microorganisms is about 1 pm. Therefore, the Raman spectrum of microorganism can be obtained at single cell level, which contains the physiological and biochemical information of microorganism cell.

[0052] (4) Only a small amount of sample is required for rapid detection. In order to surpass the detection limit of the compound, most detection methods require a sufficient number of target microorganisms to be collected from the environment. However, in combination with Raman spectroscopy and optical tweezers technology, we are able to obtain the Raman spectrum of a single cell, thereby greatly reducing the amount of sample required.

[0053] 5. Subsequently, a series of preprocessing steps are performed on the obtained Raman spectrum signal, including noise removal, background correction, signal normalization, and the application of smoothing algorithms, to improve the signal-to-noise ratio and resolution of the signal, to ensure the quality and reliability of the data. Noise removal is to eliminate random fluctuations introduced by instruments or environmental factors, and background correction is to remove non-Raman scattering signals such as fluorescence background. Through these processes, we can obtain clearer and more accurate spectral signals, and the processed Raman spectrum data will be compared with a known microbial spectrum database, which contains a large number of standard spectra of different microorganisms. Each microorganism has its unique frequency shift and intensity pattern, and the microbial species can be identified by matching the spectral characteristic peaks. This method not only reveals the presence or absence of microorganisms, but also provides information about their physiological and metabolic state. The combined application of these steps not only significantly improves the efficiency of Raman spectrum acquisition, but also greatly enhances the ability to identify and distinguish microbial cell characteristics by enhancing signal quality. In addition, the Raman spectrum data obtained by this method provides a reliable basis for subsequent quantitative analysis and biological molecule structure analysis, and has important application value in the field of biomedical research.

[0054] Although the embodiments of the present application and the drawings are disclosed for the purpose of illustration, those skilled in the art can understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, therefore, the scope of the present application is not limited to the disclosed content of the embodiments and drawings.

Claims

1. A method for detecting single bacteria based on Raman spectroscopy using DMD and microfluidics, characterized in that: The method employs a laser, a DMD (Digital Micromirror Device), a microscope, a CCD camera, a microfluidic chip, and a Raman spectroscopy detection and analysis platform. The steps of the method are as follows: S1. Collect the microbial samples to be tested and prepare the sample bacterial solution for processing in the microfluidic chip; S2. Select a microfluidic chip with a channel depth of 50μm, a width of 50μm, 100μm, and 200μm, inject the prepared bacterial solution into the microfluidic channel, construct a stable static flow field with zero flow velocity in the microfluidic chip, and confine the sample cells. The microfluidic chip is a polydimethylsiloxane (PDMS) microfluidic chip. S3. Select a laser with a wavelength of 633nm as the laser source to excite a Raman spectral signal, and the optical path enters the microfluidic chip injected with the bacterial solution to be tested. S4. After the optical path is expanded, different functions are achieved through the reflection and transmission of the optical path by the filter, and Kohler lighting is used to provide uniform and bright illumination. S5. By controlling the rotation of the micro-reflecting mirror of the DMD around a fixed axis to regulate the imaging pattern and its characteristics, the light beam is focused onto the cell to be tested, realizing the optical tweezers function, capturing the cell to be tested, and then collecting the Stokes scattering signal reflected by the cell, that is, the Raman spectrum of the microbial cell. The resolution of the micro-reflecting mirror of the DMD is 1280*800. S6. Noise removal and background correction are performed on the collected Raman spectra. By comparing with known microbial spectral databases, the types and characteristics of microorganisms are determined. S7. Based on spectral characteristics, determine the presence, type, and metabolic state of microorganisms.

2. The method for single-bacterial detection based on Raman spectroscopy using DMD and microfluidics according to claim 1, characterized in that: The filters are specific narrow-bandpass filters and long-wavepass filters.

3. The single-bacterial detection method based on Raman spectroscopy using DMD and microfluidics according to claim 1, characterized in that: The S4 uses a 600nm bandstop, a 633nm long-pass filter, and a 490nm long-pass filter to separate the reflected light path. One path is used for microscope imaging, and the other path is used for Raman spectral signal collection.

Images