A stimulated Raman microscopy device combined with optical tweezers

Abstract

The present invention belongs to the technical field of single-cell chemical imaging, and specifically is a stimulated Raman microscopy imaging device combined with optical tweezers. The device of the present invention includes a laser optical tweezers optical path, a Raman imaging optical path, and a microscope; the optical tweezers optical path couples the laser to the microscope objective through a dichroic mirror to achieve strong focusing and form a strong gradient force optical trap; the Raman optical path contains two pulsed lights that are synchronized in time domain and overlap in space. The two pulsed lasers are scanned by a scanning galvanometer and then coupled to the microscope by the optical path system to achieve two-dimensional scanning of the light beam on the sample; the optical tweezers use a position-sensitive detector to achieve real-time recording of the position of samples such as cells, and Raman imaging uses a photomultiplier tube or a photodetector after phase-locked amplification and output. The co-localized Raman light source can achieve chemical imaging of multiple components of the sample by scanning. The invented device can be used to perform Raman co-localization imaging of multiple chemical components in cells, and has important applications in the fields of cell component analysis and component distribution measurement.

Description

Technical Field

[0001] The present invention belongs to the technical field of single-cell chemical imaging, and in particular relates to a stimulated Raman microscopy imaging device. Background Art

[0002] Metabolism provides matter and energy for all life activities and is also the most basic characteristic of life. Cellular metabolism is the process of energy exchange and mutual transformation between human cells and external substances. Different cells have different metabolic cycles. For example, red blood cells need about 120 days. During the metabolic process, the composition and distribution of cells will change. How to detect changes in cell components depends on appropriate detection methods. In the 1920s, CV Raman discovered the Raman scattering effect. When photons of a specific energy are irradiated on molecules, inelastic scattering will occur. Part of the energy of the photons is absorbed by the molecules, and the scattered photons have lower energy than the incident photons. The red-shifted photons are also called Stokes photons. Raman imaging can specifically identify nucleic acids, proteins, lipids and other components in cells. The absorbed energy depends on the vibration frequency of the molecule, generally around 100cm -1 to 3500 cm -1 Raman spectroscopy can be used to distinguish chemical structures. It is both specific and robust to environmental interference. However, spontaneous Raman signals are typically weak, and obtaining images with a reliable signal-to-noise ratio typically requires a long time.

[0003] Stimulated Raman scattering (SRS) produces a stronger signal than spontaneous Raman scattering. Simultaneously shining pump light and Stokes light onto a sample stimulates stimulated Raman scattering. If the frequency difference between the two beams matches the vibrational frequency of the sample molecules, stimulated emission vibrational transitions can be generated. Stimulated Raman microscopy can be achieved by scanning the sample or the beam. The SRS signal is proportional to the component concentration, making it a powerful tool for label-free quantitative analysis of the concentration of specific chemical components.

[0004] Coherent anti-Stokes Raman scattering (CARS) is a four-wave mixing process that generates a new blue-shifted component, the anti-Stokes wave. When the energy difference matches the molecular vibration, the generated signal undergoes coherent enhancement. CARS enables damage-free, label-free molecular imaging. This is particularly important for imaging small molecules such as liposomes, as fluorescent labels can significantly affect the molecular properties. The coherent amplification caused by molecular vibrations gives CARS light excellent directionality, facilitating signal collection. The integration time of CARS is several orders of magnitude shorter than that of spontaneous Raman, while still maintaining excellent image fidelity. The CARS signal wavelength is shorter than the excitation wavelength, making it easy to spectrally separate from single-photon excited fluorescence.

[0005] Existing technologies for observing cells mostly observe them near solid substrates or under the support of biological tissues. During in vitro culture, cells are often in a nutrient solution environment and constantly change position. Real-time dynamic observation of cells is often limited by Brownian motion and imaging speed. Imaging of free cells is limited by imaging speed and the dynamic movement of cells. In particular, imaging of free cells using scanning-generated image generation technologies is difficult, mainly due to the long scanning time of a single frame and the Brownian motion of cells during the single-frame scanning time, which causes image blur.

[0006] The development of optical technology not only facilitates the observation of cell morphology but also enables contactless manipulation of cells and organelles using lasers. A strong convergence of light beams creates a strong light intensity gradient, which exerts a force on particles within it. Optical tweezers, characterized by gradient force optical traps, were invented by A. Ashkin and have found widespread application in multi-scale interdisciplinary fields, from single molecules to single cells. A. Ashkin was awarded the 2018 Nobel Prize in Physics for his invention of optical tweezers and their application in biology. Optical tweezers possess sub-piconeuron force and sub-nanometer displacement measurement accuracy, enabling them to capture cells, bacteria, and other objects.

[0007] This invention addresses the need to detect cellular components and their distribution in biomedical engineering applications. It combines single-beam gradient force optical tweezers with Raman imaging to analyze the distribution of chemical components in cells. Optical tweezers can suppress Brownian motion and reduce the image blurring effect of Raman imaging. This invention will help advance cellular studies of material and energy distribution and cellular metabolism. Summary of the Invention

[0008] The purpose of the present invention is to provide a stimulated Raman microscopy device combined with optical tweezers for chemical imaging of suspended cells, which is used to measure the spatial distribution of different components in cells and monitor cell-level metabolism.

[0009] The stimulated Raman microscopy imaging device combined with optical tweezers provided by the present invention uses laser optical tweezers to fix cells and perform Raman imaging on the cells, including laser optical tweezers, a Raman microscope, and a coupling of the two. The device can perform stimulated Raman imaging, coherent anti-Stokes imaging, or simultaneous imaging of both modes.

[0010] The laser optical tweezers include an optical tweezer laser, a high numerical aperture microscope objective, a microfluidic sample cell, a collection objective, an imaging tube lens, a position-sensitive detector, and necessary lenses and mirrors, which are sequentially connected in an optical path. The laser optical tweezers mainly use the high numerical aperture microscope objective to focus the laser beam to a diffraction-limited size, typically about 200-500 nm. Micrometer / nanometer particles near the light spot are attracted to the optical tweezers due to the light intensity gradient force.

[0011] The optical tweezers use near-infrared lasers to capture biological particles such as cells without contact and without damage, and to fix the cells without contact, providing stable position fixation for cells to achieve long-term scanning and imaging. Near-infrared lasers are generally used for non-contact and non-damage capture of biological particles such as cells, and for non-contact fixation of cells. The optical tweezers stably fix the cells without contact, greatly suppressing the image blurring effect during long scanning times.

[0012] The Raman microscope includes two pulsed lasers, a scanning mirror, a scanning lens, an imaging lens, a microscope objective lens, a collecting lens, an imaging tube lens, a filter, a photomultiplier tube, and a lock-in amplifier, etc., which are sequentially connected in an optical path; the two pulsed lasers can be synchronized in time domain and space;

[0013] The Raman microscope realizes resonance Raman imaging (SRS) or coherent anti-Stokes imaging (CARS) through the interaction of pump light and Stokes light with the sample. By adjusting the transmittance band of the filter before the photomultiplier tube, the switching between resonance Raman imaging and coherent anti-Stokes imaging can be realized. It is also possible to design a dual-channel to realize stimulated Raman imaging and coherent anti-Stokes imaging respectively.

[0014] The coupling of the laser tweezers and Raman imaging uses a dichroic mirror (such as label 110) to couple the tweezers laser and Raman excitation light to the microscope system; and then uses a dichroic mirror (such as label 116) to spatially separate the tweezers laser and Raman signal light (including stimulated Raman scattering or coherent anti-Stokes scattered light).

[0015] The laser optical tweezers are implemented using a laser whose spectrum does not overlap with the excitation light and emission light of the Raman microscope; the central wavelength of the optical tweezers can be a wavelength longer than the Stokes line, or a wavelength shorter than the anti-Stokes line, or any wavelength between the Stokes (anti-Stokes) line and the Raman excitation light.

[0016] The scanning unit of a Raman microscope consists of a scanning galvanometer, a scanning lens, an imaging lens, and a microscope objective. The scanning galvanometer performs a two-dimensional angular scan of the coaxial Raman pump and probe beams. After passing through the scanning lens, imaging lens, and microscope objective, the two spatially overlapping light spots are scanned two-dimensionally on the sample. Because the pump and probe beams overlap spatially, they can overlap spatially on the sample during the scanning process. Furthermore, a delay modulation unit introduced into one of the optical paths ensures that the two pulse trains are time-synchronized on the sample. This simultaneous temporal and spatial synchronization enables the excitation of Raman signals.

[0017] The Raman imaging is performed by scanning two spatially overlapping laser beams through a scanning unit, and after exciting the sample, stimulated Raman scattering or coherent anti-Stokes signals are collected by a photomultiplier tube, and then a two-dimensional image is reconstructed by a computer.

[0018] The optical tweezers part and the Raman microscope are respectively equipped with their own detection units.

[0019] The detection unit in the optical tweezers uses a position-sensitive detector, combined with back-focal plane interferometry, to accurately measure the position of trapped particles. Because the captured laser signal is directly detected, a neutral density attenuator is required to appropriately adjust the laser power.

[0020] The detection unit of the Raman microscope uses a highly sensitive photomultiplier tube (PMT) to detect Raman signals. Because the Raman signal is relatively weak, a lock-in amplifier (LAA) is required to amplify the signal. To achieve this, the intensity of the Raman excitation light must be modulated. This modulated signal serves as the reference input for the amplifier. Only signals from the PMT with a frequency that matches the modulation frequency of the Raman excitation light are amplified and output.

[0021] Raman imaging uses beam scanning to collect two-dimensional stimulated Raman images. Raman imaging can perform label-free, multi-component imaging of specific components in cells, such as liposomes, water, and proteins.

[0022] The present invention combines optical tweezers and Raman microscopes. During the experiment, unlabeled cells flow through the laser optical tweezers capture area through a microfluidic sample pool. When the cell to be tested is captured by the optical tweezers, the real-time position signal of the cell is recorded on the position sensitive detector, the fluid velocity in the microfluidic channel is reduced, and the signal fluctuation of the position sensitive detector gradually tends to be stable. The optical tweezers signal can be calibrated by the power spectral density. The mean square error of the position signal is then measured to ensure that the mean square error of the position signal is less than half of the Raman imaging scanning step to reduce image blur. The Raman imaging unit is then enabled to perform Raman imaging on the cell to be tested. The size of the scanning area can be adjusted according to the size of the cell to be tested, and generally needs to cover the area where the cell is captured by the optical tweezers.

[0023] The combined optical tweezers and Raman scanning imaging setup can be based on a custom-built microscope system or modified from a commercial microscope. The Raman microscope can perform stimulated Raman scattering imaging or coherent anti-Stokes imaging. Because Stokes and anti-Stokes spectral lines naturally separate in spectral space, both imaging modes can be integrated.

[0024] Advantages and features of the experimental device of the present invention:

[0025] Conventional Raman microscopes can only image samples fixed to solid substrates. For particulate samples suspended in liquids, stable images cannot be formed due to factors such as Brownian motion and the time lag of Raman scanning. Cells and other materials naturally grow in liquids or soft matter environments. Therefore, it is necessary to develop methods for non-destructive, non-contact fixation of cells and other materials. This invention uses optical tweezers to restrain cells and Raman microscopy to achieve chemical imaging.

[0026] Raman imaging microscopes combined with optical tweezers can achieve functions that neither can achieve on its own. For example, conventional Raman microscopes cannot achieve chemical imaging of cells in liquids through scanning, and optical tweezers themselves cannot perform chemical imaging of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the stimulated Raman imaging system combined with laser optical tweezers of the present invention.

[0028] Figure 2 Schematic diagram of the coherent anti-Stokes Raman imaging system combined with laser optical tweezers of the present invention.

[0029] Figure 3 Energy level diagram for stimulated Raman scattering imaging.

[0030] Figure 4 Energy level diagram for coherent anti-Stokes Raman imaging.

[0031] Figure 5 Schematic diagram of the experimental process. In the figure, the sphere represents the cell trapped by the optical tweezers. The solid line indicates the point-by-point scanning direction of stimulated Raman imaging, and the dashed line indicates when the beam switches to the next row but no signal is recorded.

[0032] Figure 6 Optical tweezers wavelength selection.

[0033] The numbers in the figure are: 101 is the first laser A, 102 is the delay and modulation unit A, 103 is the first dichroic mirror A, 104 is the second laser A, 105 is the first reflecting mirror A, 106 is the two-dimensional scanning mirror, 107 is the scanning lens A, 108 is the first imaging tube lens A, 109 is the third laser A, 110 is the second dichroic mirror A, 111 is the microscope objective lens A, 112 is the microfluidic sample pool A, 113 is the condenser lens A, 114 is the second reflecting mirror A, 115 is the second imaging tube lens A, 116 is the third dichroic mirror A, 117 is the color filter A, 118 is the photomultiplier tube A, 119 is the position sensitive detector A, and 120 is the phase-locked amplifier A.

[0034] 201 is the first laser, 202 is the delay and modulation unit, 203 is the first dichroic mirror, 204 is the two-dimensional scanning galvanometer, 205 is the second laser, 206 is the first reflector, 207 is the scanning lens, 208 is the first imaging tube lens, 209 is the illumination light source, 210 is the second reflector, 211 is the third laser, 212 is the half-wave plate, 213 is the polarization beam splitter prism, 214 is the first lens, 215 is the second dichroic mirror, 216 is the second lens, 217 is the third dichroic mirror, 218 is the first microscope objective lens, 219 is Microchannel sample pool, 220 is the second microscope objective lens, 221 is the fourth dichroic mirror, 222 is the second imaging tube lens, 223 is the first filter, 224 is the imaging camera, 225 is the fifth dichroic mirror, 226 is the third imaging tube lens, 227 is the second filter, 228 is the first photomultiplier tube, 229 is the third reflector, 230 is the relay lens, 231 is the third filter, 232 is the position sensitive detector, 233 is the third reflector, 234 is the fourth imaging tube lens, 235 is the fourth filter, and 236 is the second photomultiplier tube. DETAILED DESCRIPTION

[0035] The stimulated Raman imaging system combined with optical tweezers is described with reference to the accompanying drawings, which illustrate exemplary embodiments of the present invention and do not limit the scope of protection defined by the claims.

[0036] The present invention combines laser optical tweezers and Raman imaging in one, and can image the chemical composition of particles such as cells while optically manipulating them. The description of the embodiments is only described in conjunction with a specific type of Raman imaging. However, Raman imaging can adopt a stimulated Raman scattering imaging mode, a coherent anti-Stokes Raman imaging mode, or a combination of the two imaging modes. Existing Raman imaging microscopes can only perform chemical imaging of cells at the bottom of the glass slide. The imaging method to be achieved by the present invention can capture cells suspended in the culture medium and perform chemical imaging at the same time, which effectively ensures that the cells are completely immersed in the natural environment.

[0037] The optical tweezers part is composed of an independent laser light source that is coupled into a microscope simultaneously with a Raman imaging light source through a dichroic mirror.

[0038] The light source in the Raman imaging optical path is realized by two lasers that are synchronized in time and overlapped in space.

[0039] The Raman scanning unit is composed of a scanning galvanometer, a scanning lens, etc.

[0040] The microscope part is composed of a microscope objective lens with a high numerical aperture, a microfluidic sample pool, an imaging objective lens and the like.

[0041] The detection part includes an optical tweezers detection part and a Raman imaging detection part.

[0042] The optical tweezers detection is achieved by a position sensitive detector, and the installation position of the position sensitive detector is a position where the back focal plane of the detection objective lens is imaged through the lens.

[0043] The Raman imaging detection is collected by a photomultiplier tube and amplified and outputted by a lock-in amplifier. The modulation signal of the Raman imaging pump light is simultaneously inputted into the lock-in amplifier as a reference signal.

[0044] In the following description of the accompanying drawings, each technical feature has a preceding number indicating in which drawing the technical feature is given. The present invention will be described in detail below in conjunction with embodiments and drawings.

[0045] Example 1: A stimulated Raman imaging device combined with laser optical tweezers.

[0046] Its structure is as follows Figure 1 As shown, the apparatus includes: a first laser A101, a delay and modulation unit A102, a first dichroic mirror A103, a second laser A104, a first reflector A105, a two-dimensional scanning mirror A106, a scanning lens A107, a first imaging tube lens A108, a third laser A109, a second dichroic mirror A110, a microscope objective lens A111, a microfluidic sample cell A112, a condenser lens A113, a second reflector A114, a second imaging tube lens A115, a third dichroic mirror A116, a color filter A117, a photomultiplier tube A118, a position-sensitive detector A119, a lock-in amplifier A120, etc. This embodiment is suitable for cells without fluorescent labeling.

[0047] The first laser A101 and the second laser A104 are spatially overlapped by the first dichroic mirror A103, and the dual beams are then angularly scanned by the scanning galvanometer A106. The two wavelengths of laser light, two-dimensionally scanned by the galvanometer, are then converted into two beams for a two-dimensional scan across the sample plane after passing through the scanning lens A107, the first imaging tube lens A108, and the first microscope objective lens A111.

[0048] Raman scattering imaging can be performed using femtosecond or picosecond laser excitation, depending on the application. Femtosecond excitation utilizes a Spectra-Physics ultrafast laser (InSight X3), which outputs two synchronized beams with a fixed wavelength of 1045 nm and a tunable wavelength range of 690-1300 nm, 120 fs, and an 80 MHz repetition rate. These two synchronized dual-beam outputs enable stimulated Raman scattering imaging and coherent anti-Stokes imaging. The optical tweezers laser wavelength can be selected to be 532 nm or 650 nm, spectrally offset from the excitation and emission wavelengths used for Raman microscopy.

[0049] Picosecond dual-color lasers can be achieved using a green picosecond pump laser from NKT and an optical parametric oscillator (such as the Levante Emerald 2ps from APE). The green pump wavelength is 515 nm, and the optical parametric oscillator's wavelength is tuned between 640 and 960 nm. To avoid spectral overlap between the optical tweezers laser wavelength and the excitation and detection wavelengths used for Raman imaging, a 1064 nm or 1500 nm laser can be used for the optical tweezers laser.

[0050] The pump and Stokes pulses need to arrive at the same location on the sample simultaneously. Therefore, a delay unit is added to one of the pulse trains to adjust the time delay between the two pulse trains. Furthermore, a pair of mirrors is used to adjust the relative position of the two beams. To achieve a compact system, the intensity modulation and time delay unit A102 are integrated here.

[0051] Manipulation of micron-sized dielectric particles is achieved using single optical tweezers, while particle position is monitored using back-focal plane detection. Stimulated Raman signals or coherent anti-Stokes imaging signals are detected by photomultiplier tube A118 and output via lock-in amplifier A120. The modulation signal from light intensity modulation unit A102 serves as the reference signal for the lock-in amplification.

[0052] The optical tweezers light path originates from light source A109, is coupled to the main optical path via second dichroic mirror A110, and is focused by first microscope objective A111 to form laser optical tweezers. The capture laser light that passes forward through the sample cell is collected by second microscope objective A113, then reflected by second reflector A114, second imaging tube lens A115, and third dichroic mirror A116 before entering position-sensitive detector A119. Second imaging tube lens A115 images the back focal plane of second microscope objective A113 onto position-sensitive detector A119.

[0053] Preferably, the scanning galvanometer A 106 can use the 6200H series (Galvo) of Cambridge Technologies, the dual-axis GVS102 gold-coated two-dimensional galvanometer system of Thorlabs, or the SG1105 high-speed two-dimensional galvanometer of Jinhai Technology.

[0054] The lens and the like can be commercial devices, provided that they have a high transmittance at the corresponding wavelength and an appropriate frequency width that can cover the corresponding wavelength laser.

[0055] Reflectors can be products from any commercial company, as long as they have a high reflectivity in the corresponding bandwidth. For the visible light band, silver-coated reflectors are preferred; for the near-infrared band, metal-coated (gold, silver) reflectors are preferred; dielectric film reflectors with high reflectivity in the respective bands can also be selected.

[0056] Example 2: Coherent anti-Stokes Raman imaging device combined with laser optical tweezers.

[0057] Its structure is as follows Figure 2 As shown. It includes: a first laser 201, a delay and modulation unit 202, a first dichroic mirror 203, a two-dimensional scanning galvanometer 204, a second laser 205, a first reflector 206, a scanning lens 207, a first imaging tube lens 208, an illumination light source 209, a second reflector 210, a third laser 211, a half-wave plate 212, a polarization beam splitter prism 213, a first lens 214, a second dichroic mirror 215, a second lens 216, a third dichroic mirror 217, a first microscope objective lens 218, a microfluidic sample Sample cell 219, second microscope objective 220, fourth dichroic mirror 221, second imaging tube lens 222, first filter 223, imaging camera 224, fifth dichroic mirror 225, third imaging tube lens 226, second filter 227, first photomultiplier tube 228, sixth dichroic mirror 229, first relay lens 230, third filter 231, position sensitive detector 232, third reflector 233, fourth imaging tube lens 234, fourth filter 235, second photomultiplier tube 236.

[0058] The laser tweezers optical path begins with a third laser 211. A half-wave plate 212 and a polarizing beam-splitting prism 213 adjust the laser power. A first lens 214 and a second lens 216 expand the tweezers beam to match the back pupil size of a first microscope objective 218. The second microscope objective 220 collects the forward-scattered tweezers beam, which is then reflected by a fourth dichroic mirror 221, passes through a fifth dichroic mirror 225, and is then reflected by a sixth dichroic mirror 229 before being detected by a position-sensitive detector 232. The position-sensitive detector 232 forms an image of the back focal plane of the second microscope objective 220 via a relay lens 230.

[0059] The optical tweezers utilize an independent brightfield imaging system. Light originates from the illumination source 209, reflects off the second reflector 210 and the second dichroic mirror 215, and is then captured by the optical tweezers before entering the microscope system. The imaging light then passes through the fourth dichroic mirror 221, the second imaging tube lens 222, and the first filter 223 before being captured by the imaging camera 224.

[0060] The Raman imaging system utilizes two time-synchronized light sources: a first laser 201 and a second laser 205. Because optical pulse trains introduce time delays during spatial propagation, a delay unit is required to adjust the relative delay between the pulse trains generated by the two lasers. Furthermore, Raman detection requires a modulated signal to serve as a reference for the phase-locked detector. To maintain system compactness, both the delay and modulation functions are integrated into a single unit 202.

[0061] The first photomultiplier tube 228 detects the stimulated Raman scattering signal, while the second photomultiplier tube 236 collects the coherent anti-Stokes scattering signal. The signals from the two photomultipliers are phase-locked and amplified with the modulation signal provided by the modulation and delay module 202. Therefore, this embodiment can achieve stimulated Raman imaging and coherent anti-Stokes imaging separately, or simultaneously achieve both imaging modes.

[0062] This embodiment is applicable to cells without fluorescent labels and with various chemical compositions.

[0063] As an example, the laser can preferably be an optical parametric oscillator (OPO, Inspire) from Spectra-Physics, with a wavelength adjustment range of 345 nm to 2500 nm and a pulse width of 80-350 fs. A specific model, such as the Inspire Auto50, generates signal light tunable between 490-750 nm and idle light tunable between 930-2500 nm. The signal light serves as the pump light source for coherent anti-Stokes Raman scattering, while the idle light serves as the Stokes light. The microscope objective A106 can preferably be an Olympus 60x WIR lens with a numerical aperture of 1.2, a Nikon IR 60x lens with a NA of 1.27, or an Olympus 25x NIR lens with a numerical aperture of 1.05.

[0064] Preferably, the first photomultiplier tube 228 and the second photomultiplier tube 236 use visible light dual-alkali PMT (Thorlabs, PMM01, 280nm-630nm) or Hamamatsu's H7827 series photomultiplier tubes, such as H7827-001, whose spectral response covers 300-650nm, or photomultiplier tubes with appropriate wavelengths can be selected according to specific application requirements.

[0065] The photomultiplier tube signal is phase-locked and amplified (not shown) using a signal synchronized with the laser to detect weak anti-Stokes signals. Combined with the scanning unit 204, multi-component two-dimensional chemical imaging of particles such as cells trapped in the optical tweezers is achieved.

[0066] The relay lens 230 images the back focal plane of the second objective lens 220 onto the position sensitive detector 232. Preferably, the biological cell sample is fed into the microchannel sample pool 219 via a buffer solution, and the buffer solution is pumped to a specific flow rate via a microfluidic pump.

[0067] Preferably, the optical tweezers can use lasers with wavelengths that do not overlap with those related to Raman imaging in the frequency domain (see Figure 6(as shown), such as choosing a 1.5μm or 532nm wavelength laser. For molecules with large Raman shifts, such as proteins and nucleic acids, the optical tweezers wavelength can also be selected between the Stokes light and the Raman excitation light wavelength, or between the Raman excitation light and the anti-Stokes line.

[0068] In the detection part, the photomultiplier tube and the position sensitive detector operate in different frequency bands to reduce crosstalk. Preferably, the position sensitive detector 232 uses a Thorlabs four-quadrant detector PDQ80A with a response of 400-1050nm or a First Sensor position sensitive detector (DL100-7).

[0069] As a well-known theory, the energy level diagram of stimulated Raman scattering is as follows Figure 3 As shown in Figure 2, when pump light pumps a molecule from its ground state to a higher-energy virtual state, the molecule then transitions from the virtual state to a vibrational energy level, generating a Stokes photon. Using probe light of the same energy as the Stokes photon to excite the molecule can enhance the Stokes light, a phenomenon known as stimulated Raman scattering.

[0070] The energy level diagram of coherent anti-Stokes Raman scattering is shown in Figure 2. Figure 4 As shown, the probe light pumps the molecule from the high-energy vibrational state to the high-energy virtual state again, and the molecule jumps to the ground state again to produce high-energy photons, namely anti-Stokes photons.

[0071] In Example 1, stimulated Raman imaging can be achieved by replacing filters of different wavelength bands, or coherent Raman scattering imaging can be achieved by replacing filters.

[0072] In Example 2, the first photomultiplier tube 228 and the second photomultiplier tube 236 simultaneously detect the stimulated Raman signal and the coherent anti-Stokes Raman signal, thus achieving multimodal imaging. This combination represents a single imaging mode or a dual-modal imaging mode, and does not affect the main invention of combining optical tweezers with the detection of cellular chemical components.

[0073] Raman microscopy Figure 5 The line-by-line scanning method shown in the figure realizes two-dimensional scanning imaging. Scanned image size NxM , the obtained images are N OK, M Column. When M = N When , the image scanning area is square. Generally, it can be set to an integer power of 2 as needed, such as 256, 512, 1024, etc. Here it is implemented according to line-by-line scanning, that is, scanning the first line point by point, then scanning the second line, and so on. Estimated with a scanning accuracy of 2μm / pixel, the fluctuation of the cell position in the optical tweezers needs to be less than half a pixel size, that is, 1μm. According to the energy equipartition principle of the particle position in the optical tweezers, the lower limit of the optical trap stiffness can be estimated In order to enable the optical tweezers to capture cells stably, the optical trap stiffness needs to be adjusted to be greater than this lower limit. Or higher intensity.

[0074] This invention is directed to the collaborative operation of Raman imaging and optical tweezers. The stimulated Raman scattering imaging or coherent anti-Stokes imaging provided in the examples is used to illustrate the invention. The invention encompasses coupling a single imaging mode with optical tweezers, as well as combining two imaging modes simultaneously with optical tweezers. The goal is to provide an effective experimental method for detecting the chemical composition of particles in liquid phases such as cells. The device can also be expanded to monitor the chemical composition of particles in gas phases, such as aerosols.

Claims

1. A stimulated Raman microscopy imaging device combined with optical tweezers, characterized in that: It includes a laser optical tweezers part, a Raman microscope part and the coupling between the two; it is used for stimulated Raman imaging, or coherent anti-Stokes imaging, or simultaneous imaging of the two modes; wherein: The laser optical tweezers section includes an optical tweezers laser, a microscope objective lens, a microfluidic sample cell, a collection objective lens, an imaging tube lens, a position-sensitive detector, and necessary lenses and reflectors, which are sequentially connected by an optical path. The high numerical aperture microscope objective lens focuses the laser beam to a diffraction-limited size, specifically 200-500nm. Micrometer / nanometer particles near the light spot are attracted to the optical tweezers due to the light intensity gradient force. The optical tweezers use near-infrared lasers to capture cell biological particles without contact or damage, and to fix cells without contact, providing stable position fixation for long-term scanning and imaging of cells. The Raman microscope comprises two pulsed lasers, a scanning mirror, a scanning lens, an imaging lens, a microscope objective lens, a collecting lens, an imaging tube lens, a filter, a photomultiplier tube, and a lock-in amplifier, which are sequentially connected in an optical path. The two pulsed lasers are synchronized in time and space. The Raman microscope realizes resonance Raman imaging or coherent anti-Stokes imaging through the interaction between pump light and Stokes light and the sample; by adjusting the transmittance band of the filter before the photomultiplier tube, the switching between resonance Raman imaging and coherent anti-Stokes imaging is realized, or a dual-channel is designed to realize stimulated Raman imaging and coherent anti-Stokes imaging respectively; The coupling of laser optical tweezers and Raman imaging uses a dichroic mirror to couple the optical tweezers laser and Raman excitation light to the microscope system; then uses a dichroic mirror to spatially separate the optical tweezers laser and Raman signal light, where the Raman signal includes stimulated Raman scattering or coherent anti-Stokes scattered light. The laser optical tweezers are implemented using a laser whose spectrum does not overlap with the excitation light and emission light of the Raman microscope; the central wavelength of the optical tweezers is a wavelength longer than the Stokes line, or a wavelength shorter than the anti-Stokes line, or any wavelength between the Stokes or anti-Stokes line and the Raman excitation light; The scanning unit of the Raman microscope consists of a scanning galvanometer, scanning lens, imaging lens, and microscope objective. The scanning galvanometer performs two-dimensional angular scanning on the coaxial Raman pump light source and probe light. After passing through the scanning lens, imaging lens, and microscope objective, two-dimensional scanning is performed on the sample for the two spatially overlapping light spots. Because the pump light and probe light overlap in space, they can overlap in the sample space during the scanning process. At the same time, a delay modulation unit is introduced in one of the optical paths to ensure that the two pulse sequences are synchronized in time on the sample. This simultaneous synchronization in time and space makes it possible to excite the Raman signal. The Raman imaging is performed by scanning two spatially overlapping laser beams through a scanning unit, and after exciting the sample, the stimulated Raman scattering or coherent anti-Stokes signal is collected by a photomultiplier tube, and then a two-dimensional image is reconstructed by a computer; The optical tweezers section and the Raman microscope are each equipped with their own detection units; The position-sensitive detector in the optical tweezers serves as the detection unit, and combined with back-focal plane interferometry, it can accurately detect the position of the particles captured in the optical tweezers. Since the signal of the captured laser is directly detected, a neutral density attenuator is introduced to appropriately adjust the laser power. The Raman microscope uses a photomultiplier tube as the detection unit to detect the Raman signal. Since the intensity of the Raman signal is relatively weak, it needs to be amplified in conjunction with a phase-locked amplifier. To achieve phase-locked amplification, the intensity of the Raman excitation light needs to be modulated. The modulated signal here will serve as the reference input for the phase-locked amplification. Only the signal of the photomultiplier tube with a frequency consistent with the modulation frequency of the Raman excitation light is amplified and output.

2. The stimulated Raman microscopy imaging device combined with optical tweezers according to claim 1, characterized in that: Raman imaging uses beam scanning to collect two-dimensional stimulated Raman images. Raman imaging can perform label-free, multi-component imaging of specific components in cells, including liposomes, water, and proteins.

3. The stimulated Raman microscopy imaging device combined with optical tweezers according to claim 2, characterized in that: During the experiment, unlabeled cells flow through the laser tweezers capture area through the microfluidic sample pool; when the cell to be tested is captured by the optical tweezers, the real-time position signal of the cell is recorded on the position-sensitive detector, the fluid velocity in the microfluidic channel is reduced, and the signal fluctuation of the position-sensitive detector gradually stabilizes; the optical tweezers signal is calibrated through the power spectral density; the mean square error of the position signal is then measured to ensure that the mean square error of the position signal is less than half of the Raman imaging scanning step to reduce image blur; the Raman imaging unit is then enabled to scan the cell to be tested for Raman imaging, and the size of the scanning area is adjusted according to the size of the cell to be tested, and it needs to cover the area where the cell is captured by the optical tweezers.

4. The stimulated Raman microscopy imaging device combined with optical tweezers according to any one of claims 1 to 3, characterized in that: Specifically include: First laser A, delay and modulation unit A, first dichroic mirror A, second laser A, first reflector A, two-dimensional scanning mirror A, scanning lens A, first imaging tube lens A, third laser A, second dichroic mirror A, microscope objective lens A, microfluidic sample cell, condenser A, second reflector A, second imaging tube lens A, third dichroic mirror A, color filter A, photomultiplier tube A, position sensitive detector A, lock-in amplifier A; suitable for cells without fluorescence labeling; wherein: The first laser A and the second laser A are spatially overlapped by the first dichroic mirror A, and then the scanning mirror A performs angular scanning on the dual beams. The two wavelengths of laser light scanned two-dimensionally by the galvanometer mirror pass through the scanning lens A, the imaging tube lens A, and the first microscope objective lens A, and are converted into two beams for two-dimensional scanning on the sample plane. The pump light and Stokes light pulses need to arrive at the same position on the sample at the same time, so a delay unit is added to one of the channels to adjust the time delay between the two pulse trains. Simultaneously, a pair of mirrors is used to adjust the relative position of the two beams. The integration of the intensity modulation and time delay unit A here makes the system compact. The manipulation of micron-sized dielectric particles is achieved using single optical tweezers, and the particle position is monitored using back-focal plane detection. The stimulated Raman signal or coherent anti-Stokes imaging signal is detected by a photomultiplier tube A and then output through a lock-in amplifier A. The modulation signal of the light intensity modulation unit A serves as the reference signal for the lock-in amplifier. The optical tweezers light path starts from light source A, is coupled to the main light path through the second dichroic mirror A, and is focused by the first microscope objective lens A to form laser optical tweezers. The captured laser light that passes forward through the sample cell is collected by the second microscope objective lens A, and then reflected by the second reflecting mirror A and the second imaging tube lens A and the third dichroic mirror A, enters the position-sensitive detector A; the second imaging tube lens A images the back focal plane of the second microscope objective lens A onto the position-sensitive detector A.

5. The stimulated Raman microscopy imaging device combined with optical tweezers according to claim 4, characterized in that: Raman scattering imaging uses femtosecond or picosecond laser excitation, depending on the needs. Femtosecond excitation uses a Spectral Physics ultrafast laser that can output two synchronized beams with a fixed wavelength of 1045 nm and a tunable wavelength range of 690-1300 nm, 120 fs, and an 80 MHz repetition rate. The two synchronized dual-beam outputs can perform stimulated Raman scattering imaging and coherent anti-Stokes imaging. The optical tweezers laser wavelength is selected to be 532 nm or 650 nm to spectrally offset the excitation and emission wavelengths of Raman microscopy imaging. The picosecond dual-color laser is realized using a green picosecond pump laser and an optical parametric oscillator. The green pump wavelength is 515nm, and the optical parametric oscillator has an adjustable wavelength of 640 to 960nm. To avoid spectral overlap between the optical tweezers laser wavelength and the excitation and detection wavelengths of Raman imaging, the optical tweezers laser uses a laser with a wavelength of 1064nm or 1500nm.

6. The stimulated Raman microscopy imaging device combined with optical tweezers according to claim 5, characterized in that: Specifically include: a first laser, a delay and modulation unit, a first dichroic mirror, a two-dimensional scanning galvanometer, a second laser, a first reflector, a scanning lens, a first imaging tube lens, an illumination light source, a second reflector, a third laser, a half-wave plate, a polarization beam splitter, a first lens, a second dichroic mirror, a second lens, a third dichroic mirror, a first microscope objective lens, a microfluidic sample cell, a second microscope objective lens, a fourth dichroic mirror, a second imaging tube lens, a first filter, an imaging camera, a fifth dichroic mirror, a third imaging tube lens, a second filter, a first photomultiplier tube, a sixth dichroic mirror, a first relay lens, a third filter, a position sensitive detector, a third reflector, a fourth imaging tube lens, a fourth filter, and a second photomultiplier tube; wherein: The laser tweezers optical path begins with the third laser. A half-wave plate and polarizing beam splitter adjust the laser power. The first and second lenses expand the tweezers beam to match the back pupil size of the first microscope objective. The second microscope objective collects the forward-scattered tweezers beam, which is reflected by the fourth dichroic mirror, passes through the fifth dichroic mirror, and then reflected by the sixth dichroic mirror before being detected by a position-sensitive detector. The position sensitive detector forms an image on the rear focal plane of the second microscope objective lens through a relay lens; The optical tweezers use an independent bright-field imaging system. Starting from the illumination light source, the light is reflected by the second reflector and the second dichroic mirror, then captured by the optical tweezers and synthesized into light before entering the microscope system. The imaging light passes through the fourth dichroic mirror, the second imaging tube lens and the first filter, and is collected by the imaging camera. The Raman imaging system uses two time-synchronized light sources: the first laser and the second laser. Because optical pulse sequences cause time delays when propagating through space, a delay unit is required to adjust the relative delay between the pulse sequences generated by the two lasers. Furthermore, a modulated signal is required for the phase-locked detector to serve as a reference signal during Raman detection. To maintain system compactness, the delay and modulation functions are integrated into a single unit. The first photomultiplier tube detects the stimulated Raman scattering signal, and the second photomultiplier tube collects the coherent anti-Stokes scattering signal; the signals of the two photomultiplier tubes are phase-locked and amplified with the modulation signal provided by the modulation and delay module respectively; Stimulated Raman imaging and coherent anti-Stokes imaging can be achieved separately, or imaging of the two modes can be achieved simultaneously.

7. The stimulated Raman microscopy imaging device combined with optical tweezers according to claim 6, characterized in that: The laser uses an optical parametric oscillator from Spectral Physics, with a wavelength adjustment range of 345nm to 2500nm and a pulse width of 80-350fs; the generated signal light is tunable between 490-750nm and the idle light is tunable between 930-2500nm; the signal light is used as the pump light source for coherent anti-Stokes Raman scattering, and the idle light is used as the Stokes light.

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