Flow-type Raman sorting method and high-luminous-flux flow-type Raman sorting device

Abstract

The invention discloses a flow-type Raman sorting method and a high-luminous-flux flow-type Raman sorting device, and the method comprises the following steps: carrying out coincident focusing on Raman laser and near-infrared laser captured by a light trap to form a Raman excitation detection site, and arranging the Raman excitation detection site at the tip of the outer end of a tail end electrode in a chip dielectric focusing electrode; the photo-thermal oscillation near-infrared laser is focused on a tail end electrode in a dielectric focusing electrode of the chip and does not coincide with a Raman excitation detection site; through switching of light trap capture near-infrared laser and photo-thermal oscillation near-infrared laser, a single cell sample is captured and fixed at a Raman excitation detection site through light trap capture force generated by the light trap capture near-infrared laser or bubbles are generated through a photo-thermal effect generated by the photo-thermal oscillation near-infrared laser; the cell sample adhered to the tail end electrode in the dielectric focusing electrode is oscillated and separated through the bubbles. Compared with the prior art, the device has the beneficial effects that the organic combination of optical trap capture and dielectric capture sample oscillation functions can be realized, the switching is convenient, flexible, rapid, simple and reliable in use, the instrument cost is greatly reduced, the application range of the instrument is effectively widened, and the device has very strong universality.

Description

Technical Field

[0001] The present invention belongs to the field of flow cytometry Raman sorting, and particularly relates to a flow cytometry Raman sorting method and a high-throughput flow cytometry Raman sorting device. Background Art

[0002] A flow cytometry Raman sorter is an instrument for single-cell detection and sorting based on Raman spectroscopy. Since single cells are small, have a small component content, and are of various types, it is extremely difficult to manipulate and analyze them. Therefore, how to keep individual cells fixed for Raman signal acquisition in a flowing state and obtain a highly sensitive Raman spectrum are two of the most critical issues.

[0003] In terms of fixing individual cells, there have been implementations based on principles such as dielectric trapping and optical tweezers trapping. Dielectric trapping requires cells to be 5 - 60 μm in size, while the optical tweezers principle requires 1 - 35 μm. The applicable ranges of the two principles are different, which limits the application of these two types of devices. In particular, in dielectric trapping, a large number of cells are extremely likely to adhere to the electrodes, so certain means need to be used to oscillate and separate them to maintain the purpose of detecting and sorting individual cells. Summary of the Invention

[0004] Details of one or more embodiments of the present invention are set forth in the following drawings and description to make other features, objects, and advantages of the present application more concise and understandable.

[0005] The present invention provides a flow cytometry Raman sorting method and a high-throughput flow cytometry Raman sorting device, which can achieve the organic combination of optical tweezers trapping and dielectric trapping sample oscillation functions. When in use, the switching is convenient, flexible, fast, simple, and reliable. It not only greatly reduces the instrument cost but also effectively broadens the application range of the instrument, and has strong versatility.

[0006] The present invention discloses a flow cytometry Raman sorting method, including:

[0007] Overlapping and focusing Raman laser and near-infrared laser for optical tweezers trapping to form a Raman excitation detection site, and setting the Raman excitation detection site at the outer tip of the end electrode of the chip dielectric focusing electrode;

[0008] Focusing the near-infrared laser for optothermal oscillation on the end electrode of the chip dielectric focusing electrode without overlapping with the Raman excitation detection site;

[0009] By switching between the near-infrared laser for optical tweezers trapping and the near-infrared laser for optothermal oscillation, respectively, the optical tweezers trapping force is generated by the near-infrared laser for optical tweezers trapping to capture and fix a single-cell sample at the Raman excitation detection site, or bubbles are generated by the optothermal effect generated by the near-infrared laser for optothermal oscillation, so that the cell sample adhered to the end electrode of the chip dielectric focusing electrode is oscillated and separated through the bubbles.

[0010] In some embodiments, the distance between the light spot formed by the optothermal oscillating near-infrared laser on the end electrode in the dielectric focusing electrode and the Raman excitation detection site is 5-15 μm.

[0011] In some embodiments, both the optothermal oscillating near-infrared laser and the Raman excitation detection site are located on the central axis of the tip of the end electrode in the dielectric focusing electrode.

[0012] The present invention also discloses a near-infrared optical path for a flow cytometry Raman sorting device, including:

[0013] A near-infrared laser;

[0014] A polarization beam splitting prism, configured to receive the near-infrared laser emitted by the near-infrared laser and split it into an optical trap capture near-infrared laser optical path and an optothermal oscillating near-infrared laser optical path;

[0015] A shutter and a Galilean beam expander assembly, provided on the optical trap capture near-infrared laser optical path and the optothermal oscillating near-infrared laser optical path;

[0016] An optical path adjustment assembly, configured to receive the optical trap capture near-infrared laser optical path and the optothermal oscillating near-infrared laser optical path and, by adjustment, make the light spots formed by the optical trap capture near-infrared laser optical path and the optothermal oscillating near-infrared laser optical path on the end electrode of the chip dielectric focusing electrode not coincide, so as to respectively form an optical trap capture near-infrared laser acting on the outer tip of the end electrode of the chip dielectric focusing electrode to generate an optical trap capture force and an optothermal oscillating near-infrared laser acting on the end electrode of the chip dielectric focusing electrode to generate a bubble oscillation force through the optothermal effect.

[0017] In some embodiments, the optical path adjustment assembly includes:

[0018] A transmissive and reflective beam displacement flat plate, arranged in an inclined structure and coaxially arranged with the polarization beam splitting prism and the optothermal oscillating near-infrared laser optical path;

[0019] A first mirror and a second mirror, provided on the optical trap capture near-infrared laser optical path; the first mirror is configured to reflect the optical trap capture near-infrared laser optical path emitted by the polarization beam splitting prism; the second mirror is configured to reflect the optical trap capture near-infrared laser optical path reflected by the first mirror onto the transmissive and reflective beam displacement flat plate;

[0020] A light spot reflection adjustment mirror, configured to receive the optical trap capture near-infrared laser optical path formed by reflection through the transmissive and reflective beam displacement flat plate or the optothermal oscillating near-infrared laser optical path formed by transmission through the transmissive and reflective beam displacement flat plate.

[0021] The present invention also discloses a high-flux flow Raman sorting device, including:

[0022] A three-axis displacement stage, with a visible light microscope objective for receiving the Raman signal acquisition and detection optical path and a near-infrared microscope objective for receiving the near-infrared optical path as claimed or described respectively above and below the three-axis displacement stage;

[0023] A Raman excitation optical path, a microscopic imaging optical path, and a Köhler coaxial illumination optical path, which are arranged along the Raman signal acquisition and detection optical path and converge into the Raman signal acquisition and detection optical path through a low-beam Raman filter a, a thin-film beam splitter, and a flat beam splitter respectively;

[0024] The thin-film beam splitter and the flat beam splitter can be deflected, and by deflection, the microscopic imaging optical path and the Köhler coaxial illumination optical path are converged into the Raman signal acquisition and detection optical path or do not obstruct the Raman signal acquisition and detection optical path.

[0025] In some embodiments, the Raman excitation optical path, the microscopic imaging optical path, and the Köhler coaxial illumination optical path are arranged in a parallel structure.

[0026] In some embodiments, the Raman signal acquisition and detection optical path includes a conjugate mechanical spatial filtering component, an off-axis aspherical mirror, a low-beam Raman filter b, a relay optical path component, and a spectrometer;

[0027] The Raman excitation optical path includes a Raman single longitudinal mode laser, a Raman Galilean beam expander component, and a conjugate Rayleigh line filtering component;

[0028] The microscopic imaging optical path includes a high-definition camera, a notch filter component, and an imaging lens;

[0029] The Köhler coaxial illumination optical path includes an LED and a Köhler coaxial illumination component.

[0030] The present invention also discloses a sorting method based on the high-flux flow Raman sorting device, including a dielectric capture flow Raman sorting method and an optical trap capture flow Raman sorting method;

[0031] The dielectric capture flow Raman sorting method is as follows:

[0032] Load the cell sample by pneumatic drive, rotate the thin-film spectroscope and the flat spectroscope to converge the microscopic imaging optical path and the Köhler coaxial illumination optical path into the Raman signal acquisition and detection optical path to observe the cell state in real time, connect a periodic dielectric signal to the dielectric focusing electrode, and control the flow rate of the cell sample by controlling the dielectric on-off timing and adjusting the pneumatic drive pressure for sample loading, so that the cell sample passes through the dielectric focusing electrode for focusing, so as to achieve that a single cell focuses and flows through and is captured at the outer tip of the end electrode in the dielectric focusing electrode, that is, the Raman excitation detection site;

[0033] Turn on the Raman excitation optical path to emit a Raman excitation source, focus the cell sample at the outer tip of the end electrode in the dielectric focusing electrode through a visible light microscope objective lens, the generated Raman signal enters the Raman signal acquisition and detection optical path, and passes through the thin-film spectroscope and the flat spectroscope to make it not block the Raman signal acquisition and detection optical path, thereby forming a single-cell Raman spectrum to provide a criterion for sorting;

[0034] Judge and identify through the host computer, and sort the cells by connecting a periodic dielectric signal to the dielectric sorting electrode;

[0035] During the above process, keep the optical trap capture near-infrared laser optical path closed, intermittently turn on the optothermal oscillation near-infrared laser optical path, adjust the position of the optothermal oscillation near-infrared laser through the optical path adjustment component, and generate a bubble oscillation force through the optothermal effect to oscillate and separate the cells adhered to the outer tip of the end electrode in the dielectric focusing electrode;

[0036] The optical trap capture flow-through Raman sorting method is as follows:

[0037] Load the cell sample by pneumatic drive, rotate the thin-film spectroscope and the flat spectroscope to converge the microscopic imaging optical path and the Köhler coaxial illumination optical path into the Raman signal acquisition and detection optical path to observe the cell state in real time, introduce a double-layer sheath fluid into the chip to form a sheath flow, adjust the pneumatic drive pressure for sample loading to control the sample flow rate and the sheath fluid flow rate, and finally make the cell sample form a single-cell pipeline in the central flow field passing through the Raman excitation detection site in the chip;

[0038] Turn on the optical trap capture near-infrared laser optical path, capture the cell sample at the Raman excitation detection site with the optical trap capture near-infrared laser, and simultaneously pause the pneumatic drive pressure for sample loading;

[0039] Turn on the Raman excitation optical path to emit a Raman excitation source, focus the cell sample at the outer tip of the end electrode in the dielectric focusing electrode through a visible light microscope objective lens, the generated Raman signal enters the Raman signal acquisition and detection optical path, and passes through the thin-film spectroscope and the flat spectroscope to make it not block the Raman signal acquisition and detection optical path, thereby forming a single-cell Raman spectrum to provide a criterion for sorting;

[0040] It is judged and recognized by the host computer, and the cell sample captured by the optical trap is sorted by moving the three-axis displacement stage.

[0041] In some embodiments, the applicable cell sample size in the dielectric capture flow Raman sorting method is 5 - 60 μm; the applicable cell sample size in the optical trap capture flow Raman sorting method is less than 5 μm.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] 1. By combining the dielectric microfluidic chip, the near-infrared laser for optical trap capture and the near-infrared laser for photothermal oscillation are set, so as to respectively capture and fix a single cell sample at the Raman excitation detection site through the optical trap capture force generated by the near-infrared laser for optical trap capture, or generate bubbles through the photothermal effect generated by the near-infrared laser for photothermal oscillation, so that the cell sample adhered to the end electrode in the dielectric focusing electrode is oscillated and separated through the bubbles, thereby realizing the organic combination of optical trap capture and dielectric capture (including the sample oscillation function), not only greatly reducing the instrument cost, but also effectively broadening the application range of the instrument, and having strong versatility.

[0044] 2. A near-infrared optical path is designed by combining optical trap capture and dielectric capture (including the sample oscillation function). Through a single near-infrared laser, the organic combination of optical trap capture and dielectric capture (including the sample oscillation function) can be realized simultaneously. When in use, it is convenient to switch, flexible and fast, simple and reliable.

[0045] 3. An optical path structure based on common optical path switching is designed. On the one hand, the excitation light can be irradiated on the sample efficiently and without loss; on the other hand, the returned Raman signal can return to the Raman signal acquisition and detection optical path with as little interference as possible and as high a light flux as possible, and the number of optical elements passed through during the Raman signal acquisition and detection process is minimized, realizing small signal loss and short acquisition time, and finally achieving the purpose of high light flux measurement. Through experimental verification, compared with the commercial microconfocal Raman spectroscopy measurement system, the Raman excitation light flux is increased from generally 50% to 85%, and the Raman signal collection light flux is increased from generally not higher than 30% to 41.8%, and the collection light flux is increased by about 40%, realizing the detection of high light flux living single cells.

[0046] 4. The overall optical path of the device adopts a coaxial common optical path design, with a planar layout and all optical components placed perpendicular or at 45° to the optical axis, without non-standard angles, making each optical path relatively independent and easy to debug, the system integration is flexible and simple, greatly reducing the system debugging and maintenance cost, the optical path layout has a reasonable turn, the overall solidification degree of the device is high, the anti-vibration and shock resistance ability is strong, and the stability is high. Description of the Drawings

[0047] The accompanying drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0048] Figure 1 It is a schematic diagram of the distribution structure of the optical trap for capturing near-infrared laser and the optothermal oscillation infrared laser of the present invention.

[0049] Figure 2 It is a schematic diagram of the structure of the sorting device of the present invention.

[0050] Description of the drawings: Raman laser 1, optical trap for capturing near-infrared laser 2, optothermal oscillation infrared laser 3, near-infrared laser 4, polarization beam splitter prism 5, shutter 6, Galilean beam expander assembly 7, transmissive-reflective beam displacement flat 8, spot reflection adjustment mirror 9, near-infrared microscope objective 10, three-axis displacement stage 11, visible light microscope objective 12, flat beam splitter 13, thin film beam splitter 14, LED 15, Köhler coaxial illumination assembly 16, high-definition camera 17, notch filter assembly 18, imaging lens 19, Raman single longitudinal mode laser 20, Raman Galilean beam expander assembly 21, conjugate Rayleigh line filtering assembly 22, low beam Raman filter a 23, conjugate mechanical spatial filtering assembly 24, off-axis aspherical mirror 25, low beam Raman filter b 26, relay optical path assembly 27, spectrometer 28. Detailed implementation manners

[0051] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described and explained below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments provided by the present invention without creative efforts fall within the scope of protection of the present invention.

[0052] Obviously, the accompanying drawings in the following description are only some examples or embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, the present invention can also be applied to other similar scenarios based on these drawings. In addition, it can also be understood that although the efforts made in this development process may be complex and lengthy, for those of ordinary skill in the art related to the content disclosed by the present invention, some design, manufacturing or production changes based on the technical content disclosed by the present invention are only conventional technical means and should not be understood as the content disclosed by the present invention being insufficient.

[0053] Reference to "embodiments" in the present invention means that specific features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of the present invention. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment each time, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. It is explicitly and implicitly understood by those of ordinary skill in the art that the embodiments described in the present invention can be combined with other embodiments without conflict.

[0054] A flow cytometry Raman sorting method, which is specifically implemented in combination with a dielectric microfluidic chip. The dielectric microfluidic chip includes a dielectric focusing electrode and a sorting electrode. The specific structural forms of the dielectric focusing electrode and the sorting electrode are as Figure 1 shown. Since the above structure is prior art, it will not be elaborated in detail. The method is specifically as follows: The Raman laser 1 and the optical trap capture near-infrared laser 2 are overlapped and focused to form a Raman excitation detection site, and the Raman excitation detection site is set at the outer tip of the end electrode in the dielectric focusing electrode of the chip, that is, Figure 1 at the outer tip of the V-shaped structure electrode at the rightmost end of the dielectric focusing electrode; the photothermal oscillation near-infrared laser 3 is focused on the end electrode in the dielectric focusing electrode of the chip and does not overlap with the Raman excitation detection site; by switching between the optical trap capture near-infrared laser 2 and the photothermal oscillation near-infrared laser 3, the optical trap capture force generated by the optical trap capture near-infrared laser 2 is used to capture and fix a single cell sample at the Raman excitation detection site respectively, or bubbles are generated by the photothermal effect generated by the photothermal oscillation near-infrared laser 3, so that the cell sample adhered to the end electrode in the dielectric focusing electrode is oscillated and separated through the bubbles.

[0055] Since the end electrode in the dielectric focusing electrode of the chip needs to achieve both optical trap capture and photothermal oscillation separation, when setting, the width of the end electrode in the dielectric focusing electrode of the chip can be considered to be set larger than the width of the electrodes in other dielectric focusing electrodes of the chip.

[0056] Specifically, the distance between the light spot formed by the photothermal oscillation near-infrared laser 3 on the end electrode in the dielectric focusing electrode and the Raman excitation detection site is 5 - 15 μm. Considering the actual size problem and avoiding focusing of the near-infrared laser on the same point, which is likely to cause the problem that the thermal energy caused by the photothermal effect cannot be quickly discharged within the narrow area of the electrode, and the electrode tip will be directly broken down and damaged. Therefore, a misaligned and non-overlapping setting is adopted to reduce the probability of the above problem. Preferably, the specific distance is set to 8 - 12 μm.

[0057] Specifically, both the photothermal oscillation near-infrared laser 3 and the Raman excitation detection site are located on the central axis of the tip of the middle and end electrodes of the dielectric focusing electrode. During the actual sorting process, under the influence of the dielectrophoretic force, the cells will flow along the direction of the electrode tip, and the above settings can make the photothermal oscillation effect better.

[0058] A near-infrared light path for a flow-through Raman sorting device, applicable to the above-mentioned flow-through Raman sorting method. Specifically, this near-infrared light path provides the corresponding optical trap to capture the near-infrared laser 2 and the photothermal oscillation near-infrared laser 3. Specifically, it includes:

[0059] A near-infrared laser 4, serving as the laser source for cell sample oscillation and optical trap capture. Its wavelength can be in the near-infrared band such as 785 nm, 1064 nm, etc., and it needs to have a high power (>500 mW), a narrow line width (<1 MHz), and a high stability (<5%);

[0060] A polarization beam splitting prism 5, used to receive the near-infrared laser emitted by the near-infrared laser 4 and split it into an optical trap capture near-infrared laser light path and a photothermal oscillation near-infrared laser light path;

[0061] A shutter 6 and a Galilean beam expander 7 are provided on the optical trap capture near-infrared laser light path and the photothermal oscillation near-infrared laser light path; the shutter 6 is used to control the switching of the opening and closing of the corresponding light paths respectively, and the Galilean beam expander 7 is used to collimate and expand the light path to an appropriate aperture. Its beam expansion ratio is limited to 2:1 - 7:1 according to the different entrance pupils of the near-infrared microscope objective, and the spot size of the photothermal oscillation near-infrared laser light path on the middle and end electrodes of the dielectric focusing electrode is adjusted as needed.

[0062] An optical path adjustment component, used to receive the optical trap capture near-infrared laser light path and the photothermal oscillation near-infrared laser light path and make the spots formed by the optical trap capture near-infrared laser light path and the photothermal oscillation near-infrared laser light path on the middle and end electrodes of the chip dielectric focusing electrode not coincide by adjustment, so as to respectively form the optical trap capture near-infrared laser 2 acting on the outer tip of the middle and end electrodes of the chip dielectric focusing electrode to generate the optical trap capture force and the photothermal oscillation near-infrared laser 3 acting on the middle and end electrodes of the chip dielectric focusing electrode to generate the bubble oscillation force through the photothermal effect. The optical path adjustment component ensures that the distance between the spot formed by the photothermal oscillation near-infrared laser 3 on the middle and end electrodes of the dielectric focusing electrode and the Raman excitation detection site is within the range of 5 - 15 μm.

[0063] Specifically, the optical path adjustment component includes:

[0064] A transmissive and reflective beam displacement flat plate 8, which is arranged in an inclined structure and is coaxially arranged with the polarization beam splitting prism 5 and the photothermal oscillation near-infrared laser light path. Specifically, the set inclination angle is 45°;

[0065] The first reflector and the second reflector are arranged on the optical path of the near-infrared laser for optical trap capture; the first reflector is used to reflect the optical path of the near-infrared laser for optical trap capture emitted by the polarization beam splitter prism 5, and is specifically arranged at an inclined 45°; the second reflector is used to reflect the optical path of the near-infrared laser for optical trap capture reflected by the first reflector onto the transmissive-reflective beam displacement flat plate 8, and is specifically arranged at an inclined 45°.

[0066] The spot reflection adjustment mirror 9 is used to receive the optical path of the near-infrared laser for optical trap capture formed by reflection through the transmissive-reflective beam displacement flat plate 8 or the optical path of the near-infrared laser for photothermal oscillation formed by transmission through the transmissive-reflective beam displacement flat plate 8, and is specifically arranged at an inclined 45°.

[0067] In the above adjustment assembly, due to the characteristics of its own structure and belonging to a light-transmitting component, and having a certain thickness, for example, through the setting method of its inclined structure, the optical path of the near-infrared laser for photothermal oscillation after transmission and the optical path of the near-infrared laser for optical trap capture after refraction will have a positional deviation, and this positional deviation is exactly applied to the positional deviation of the final near-infrared laser 2 for optical trap capture and the near-infrared laser 3 for photothermal oscillation on the end electrodes of the chip dielectric focusing electrode. The specific setting method of the error size can be achieved by replacing the thickness of the transmissive-reflective beam displacement flat plate 8. The setting form of its thickness and the finally formed spacing of 5 - 15 μm can be obtained through conventional comparison calculations, so it will not be elaborated here.

[0068] Specifically, the optical path of the near-infrared laser for optical trap capture is formed by reflection through the polarization beam splitter prism 5, and its path sequentially passes through the shutter 6, the reflection of the first reflector, the Galilean beam expander assembly 7, the reflection of the second reflector, the reflection of the transmissive-reflective beam displacement flat plate 8, and the reflection of the spot reflection adjustment mirror 9; the optical path of the near-infrared laser for photothermal oscillation is formed by transmission through the polarization beam splitter prism 5, and its path sequentially passes through the shutter 6, the Galilean beam expander assembly 7, the transmission of the transmissive-reflective beam displacement flat plate 8, and the reflection of the spot reflection adjustment mirror 9.

[0069] A high-flux flow cytometry Raman sorting device includes:

[0070] A three-axis displacement stage 11, and a visible light microscope objective 12 for receiving the Raman signal acquisition and detection optical path and a near-infrared microscope objective 10 for receiving the near-infrared optical path as described above are respectively provided above and below the three-axis displacement stage 11;

[0071] The Raman excitation optical path, the microscopic imaging optical path, and the Köhler coaxial illumination optical path are arranged along the Raman signal acquisition and detection optical path, and respectively converge into the Raman signal acquisition and detection optical path through the low-beam Raman filter a23, the thin film beam splitter 14, and the flat beam splitter 13;

[0072] The thin-film beam splitter 14 and the planar beam splitter 13 can be deflected so that the microscopic imaging optical path and the Köhler coaxial illumination optical path converge into the Raman signal acquisition and detection optical path or do not obstruct the Raman signal acquisition and detection optical path through deflection.

[0073] Specifically, by setting the thin-film beam splitter 14 and the planar beam splitter 13 as deflectable structures, when they are in the 45-degree state, the microscopic imaging optical path and the Köhler coaxial illumination optical path can be converged into the Raman signal acquisition and detection optical path through reflection; when they are in the 90-degree state, the thin-film beam splitter 14 and the planar beam splitter 13 completely leave the Raman signal acquisition and detection optical path, thus eliminating the signal splitting loss due to the coaxial common optical path and performing high-throughput Raman signal acquisition.

[0074] In some embodiments, the Raman excitation optical path, the microscopic imaging optical path, and the Köhler coaxial illumination optical path are arranged in a parallel structure.

[0075] Specifically, the Raman signal acquisition and detection optical path includes a conjugate mechanical spatial filtering component 24, an off-axis aspherical mirror 25, a low-beam Raman filter b26, a relay optical path component 27, and a spectrometer 28; the Raman signal acquisition and detection optical path is the core optical path of this device, and its function is to collect and detect Raman signals with high throughput. The thin-film beam splitter 14 and the planar beam splitter 13 are used for common optical path switching. Specifically, the planar beam splitter and the thin-film beam splitter are rotated from the 45-degree state to the 90-degree state simultaneously or non-simultaneously, so as to completely separate from the Raman signal acquisition optical path, thus eliminating the signal splitting loss due to the coaxial common optical path and performing high-throughput Raman signal acquisition. Its characteristics are high speed (each switching < 1 s) and high repeatability (repeat positioning accuracy < 0.5 μm); the low-beam Raman filter a23 is used for high-throughput transmission of Raman signals; the conjugate mechanical spatial filtering component 24 functions to achieve true confocal imaging of microscopic Raman. Its characteristic is that the pinhole disk in the component can be controlled to rotate by an optical encoding motor, and pinholes of different sizes can be rotated into the optical path as needed, so as to prevent unnecessary signals outside the microscope focal plane from reaching the detector, playing a role in spatial filtering and improving the spatial resolution of the instrument (< 1.5 μm); the off-axis aspherical mirror 25 is used for collimating and turning the Raman signal; the low-beam Raman filter b26 is used for further filtering out Rayleigh signals and highly transmitting Raman signals. Its characteristics are ultra-high edge steepness (0.2%), ultra-low wave number (< 50 cm-1), high Rayleigh cut-off rate (OD > 6), and high Raman signal transmittance (> 93%); the relay optical path component 27 is used for imaging the Raman signal onto the slit of the spectral monochromator with high NA matching; the reflector is used for turning the optical path; the spectrometer 28 is used for splitting and detecting the Raman signal, and finally transmitting it to the image workstation to form a single-cell Raman spectrum, thus providing a criterion for sorting.

[0076] The Raman excitation optical path includes a Raman single longitudinal mode laser 20, a Raman Galilean beam expander assembly 21, and a conjugate Rayleigh line filter assembly 22. Specifically, the function of the Raman excitation optical path is to provide a Raman signal excitation source with narrow linewidth and high stability. It includes: The Raman single longitudinal mode laser 20 is a laser source for exciting Raman signals, and its wavelength can be in the visible light band such as 514.5 nm, 532 nm, 632.8 nm, etc. It is characterized by high power (>200 mW), narrow linewidth (<1 MHz), single longitudinal mode (M2 < 1.1), and high stability (<2%). These characteristics ensure that the laser spot has a very high spatial resolution (<0.6 μm) after microscopic imaging; The Raman Galilean beam expander assembly 21 is used to collimate and expand the laser to an appropriate aperture, and its beam expansion ratio is limited to 2:1 - 7:1 according to the different entrance pupils of the visible light microscopic objective lens; The conjugate Rayleigh line filter assembly 22 is used to filter out laser wavelengths other than Rayleigh light; The low beam Raman filter a23 is used to highly reflect the excitation light and highly cut off the returned Rayleigh light, so as to highly transmit the Raman signal. It is characterized by a high edge steepness (0.5%), a low wave number (<100 cm-1), a high excitation light reflectivity (>94%), and a high Raman signal transmittance (>93%). Corresponding mirrors can be set in the optical path for folding the optical path.

[0077] The microscopic imaging optical path includes a high-definition camera 17, a notch filter assembly 18, and an imaging lens 19. The function of the microscopic imaging optical path is to provide a visual cell morphology and spatial position for accurate measurement of "seeing is believing". It is characterized by an infinity microscopic imaging system. The visible light microscopic objective lens 12 is used for microscopic imaging of samples and Raman signal excitation. It is characterized by high magnification (>50X), high NA (>0.8), ultra-flat field, and apochromatic; The three-axis displacement stage 11 adopts a high-precision structure to move the chip with the built-in sample. It is characterized by precise movement of the chip in the front, back, left, right, up, and down directions through DC motor control. The minimum step value of the movement is 20 nm, the repeat positioning accuracy during the movement is 0.5 μm, and the movement range is ±20 mm in the front and back, ±35 mm in the left and right, and ±20 mm in the up and down; The thin film beam splitter 14 is used to reflect the sample image coaxial and co-optical path onto the imaging lens. It is characterized by a very small optical path change (<5 μm), almost no ghosting, and no chromatic aberration in the focused beam; The imaging lens 19 is used to focus the sample image onto the high-definition color camera. It is characterized by wide-field imaging, apochromatic, and achieving diffraction limit across the entire field of view; The notch filter assembly 18 is used to filter out Rayleigh light and near-infrared light to enable clear imaging and protect the photosensitive surface of the camera. It is characterized by providing high OD cut-off (OD > 6) for Rayleigh light and near-infrared light respectively; The high-definition camera 17 is used for sample imaging. It is characterized by a large rake face (≥1 inch) and high resolution (>12 million pixels). Corresponding mirrors can be set in the optical path for folding the optical path.

[0078] The Köhler coaxial illumination optical path includes LED15 and the Köhler coaxial illumination component 16. The Köhler coaxial illumination optical path provides suitable bright-field illumination for microscopic imaging, and the white light LED lamp provides the illumination light source; the Köhler coaxial illumination component is designed based on the Köhler illumination principle to provide a light field with high uniformity; the flat beam splitter 13 is used to coaxially introduce the illumination light source into the microscopic objective lens along the common optical path.

[0079] A sorting method based on the above high-flux flow Raman sorting device includes a dielectric capture flow Raman sorting method and an optical trap capture flow Raman sorting method;

[0080] The dielectric capture flow Raman sorting method is as follows:

[0081] The cell sample is loaded by pneumatic drive, the rotating thin film beam splitter 14 and the flat beam splitter 13 are rotated to converge the microscopic imaging optical path and the Köhler coaxial illumination optical path into the Raman signal acquisition and detection optical path to observe the cell state in real time, and a periodic dielectric signal is applied to the dielectric focusing electrode. By controlling the dielectric on-off timing and adjusting the loading drive air pressure to control the flow rate of the cell sample, the cell sample passes through the dielectric focusing electrode for focusing, so as to realize that a single cell focuses and flows through and is captured at the outer tip of the end electrode in the dielectric focusing electrode, that is, the Raman excitation detection site;

[0082] The Raman excitation optical path is turned on to emit a Raman excitation source, and the cell sample at the outer tip of the end electrode in the dielectric focusing electrode is focused by the visible light microscopic objective lens 12. The generated Raman signal enters the Raman signal acquisition and detection optical path, and the rotating thin film beam splitter 14 and the flat beam splitter 13 are rotated to make them not block the Raman signal acquisition and detection optical path, so as to form a single-cell Raman spectrum to provide a criterion for sorting;

[0083] Through the judgment and recognition of the host computer, the cells are sorted by applying a periodic dielectric signal to the dielectric sorting electrode;

[0084] In the above process, the optical trap capture near-infrared laser optical path is kept closed, and the photo-thermal oscillation near-infrared laser optical path is intermittently turned on. The position of the photo-thermal oscillation near-infrared laser 3 is adjusted by adjusting the optical path adjustment component, and a bubble oscillation force is generated through the photo-thermal effect to oscillate and separate the cells adhered to the outer tip of the end electrode in the dielectric focusing electrode;

[0085] The optical trap capture flow Raman sorting method is as follows:

[0086] The cell sample is loaded by air pressure drive. The thin film spectroscope 14 and the flat spectroscope 13 are rotated to converge the microscopic imaging optical path and the Köhler coaxial illumination optical path into the Raman signal acquisition and detection optical path to observe the cell state in real time. A double-layer sheath fluid is introduced into the chip to form a sheath flow. The air pressure of the loading drive is adjusted to control the sample flow rate and the sheath fluid flow rate. Finally, the cell sample forms a single-cell pipeline in the central flow field passing through the Raman excitation detection site in the chip;

[0087] The optical trap captures the near-infrared laser optical path. The cell sample at the Raman excitation detection site is captured by the near-infrared laser 2 of the optical trap, and at the same time, the air pressure of the loading drive is paused;

[0088] The Raman excitation optical path is turned on to emit the Raman excitation source. The cell sample at the outer tip of the end electrode in the dielectric focusing electrode is focused by the visible light microscope objective 12. The generated Raman signal enters the Raman signal acquisition and detection optical path, and through the rotation of the thin film spectroscope 14 and the flat spectroscope 13, it does not obstruct the Raman signal acquisition and detection optical path, thereby forming a single-cell Raman spectrum to provide a criterion for sorting;

[0089] Through the judgment and recognition of the host computer, the cell sample captured by the optical trap is sorted by moving the three-axis displacement stage 11.

[0090] Specifically, the applicable cell sample size in the dielectric capture flow Raman sorting method is 5 - 60 μm; the applicable cell sample size in the optical trap capture flow Raman sorting method is less than 5 μm.

[0091] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A flow cytometry Raman sorting method, characterized in that, Comprising: Coincidentally focusing the Raman laser and the near-infrared laser for optical trap capture to form a Raman excitation detection site, and setting the Raman excitation detection site at the outer tip of the middle end electrode of the chip dielectric focusing electrode; Focusing the near-infrared laser for optothermal oscillation on the middle end electrode of the chip dielectric focusing electrode without coinciding with the Raman excitation detection site; By switching the near-infrared laser for optical trap capture and the near-infrared laser for optothermal oscillation, respectively, the optical trap capture force generated by the near-infrared laser for optical trap capture is used to capture and fix a single cell sample at the Raman excitation detection site, or the optothermal effect generated by the near-infrared laser for optothermal oscillation emits bubbles to oscillate and separate the cell sample adhered to the middle end electrode of the dielectric focusing electrode through the bubbles.

2. The sorting method according to claim 1, characterized in that, The distance between the light spot formed by the near-infrared laser for optothermal oscillation on the middle end electrode of the dielectric focusing electrode and the Raman excitation detection site is 5 - 15 μm.

3. The sorting method according to claim 1, wherein Both the near-infrared laser for optothermal oscillation and the Raman excitation detection site are located on the tip central axis of the middle end electrode of the dielectric focusing electrode.

4. A near-infrared optical path for a flow cytometry Raman sorting device, characterized in that, Comprising: A near-infrared laser; A polarization beam splitting prism, which is used to receive the near-infrared laser emitted by the near-infrared laser and split it into an optical trap capture near-infrared laser optical path and an optothermal oscillation near-infrared laser optical path; A shutter and a Galilean beam expander assembly, which are arranged on the optical trap capture near-infrared laser optical path and the optothermal oscillation near-infrared laser optical path; An optical path adjustment assembly, which is used to receive the optical trap capture near-infrared laser optical path and the optothermal oscillation near-infrared laser optical path and, through adjustment, make the light spots formed by the optical trap capture near-infrared laser optical path and the optothermal oscillation near-infrared laser optical path on the middle end electrode of the chip dielectric focusing electrode not coincide, so as to respectively form an optical trap capture near-infrared laser acting on the outer tip of the middle end electrode of the chip dielectric focusing electrode to generate an optical trap capture force and an optothermal oscillation near-infrared laser acting on the middle end electrode of the chip dielectric focusing electrode to generate a bubble oscillation force through the optothermal effect.

5. The near-infrared light path according to claim 4, wherein The optical path adjustment assembly includes: A transmissive-reflective beam displacement flat plate, which is arranged in an inclined structure and coaxially arranged with the polarization beam splitting prism and the optothermal oscillation near-infrared laser optical path; A first reflecting mirror and a second reflecting mirror, which are arranged on the optical trap capture near-infrared laser optical path; the first reflecting mirror is used to reflect the optical trap capture near-infrared laser optical path emitted by the polarization beam splitting prism; the second reflecting mirror is used to reflect the optical trap capture near-infrared laser optical path reflected by the first reflecting mirror onto the transmissive-reflective beam displacement flat plate; A light spot reflection adjustment mirror, which is used to receive the optical trap capture near-infrared laser optical path formed by reflection through the transmissive-reflective beam displacement flat plate or the optothermal oscillation near-infrared laser optical path formed by transmission through the transmissive-reflective beam displacement flat plate.

6. A high-light-flux flow cytometry Raman sorting device, characterized in that, Comprising: A three-axis displacement stage, and a visible light microscope objective for receiving the Raman signal acquisition and detection optical path and a near-infrared microscope objective for receiving the near-infrared optical path as described in claim 4 or 5 are respectively arranged above and below the three-axis displacement stage; The Raman excitation optical path, the microscopic imaging optical path, and the Köhler coaxial illumination optical path are arranged through the Raman signal acquisition and detection optical path, and are respectively converged into the Raman signal acquisition and detection optical path through the low-beam Raman filter a, the thin-film beam splitter, and the flat beam splitter; The thin-film beam splitter and the flat beam splitter can be deflected, and by deflection, the microscopic imaging optical path and the Köhler coaxial illumination optical path are converged into the Raman signal acquisition and detection optical path or do not obstruct the Raman signal acquisition and detection optical path.

7. The high-flux flow cytometry Raman sorting device according to claim 6, characterized in that, The Raman excitation optical path, the microscopic imaging optical path, and the Köhler coaxial illumination optical path are arranged in a parallel structure.

8. The high-throughput flow Raman sorting device according to claim 6, wherein The Raman signal acquisition and detection optical path includes a conjugate mechanical spatial filtering component, an off-axis aspherical mirror, a low-beam Raman filter b, a relay optical path component, and a spectrometer; The Raman excitation optical path includes a Raman single longitudinal mode laser, a Raman Galilean beam expander component, and a conjugate Rayleigh line filtering component; The microscopic imaging optical path includes a high-definition camera, a notch filter component, and an imaging lens; The Köhler coaxial illumination optical path includes an LED and a Köhler coaxial illumination component.

9. The sorting method of the high-light-flux flow cytometry Raman sorting device according to claim 6, characterized in that, It includes a dielectric capture flow Raman sorting method and an optical trap capture flow Raman sorting method; The dielectric capture flow Raman sorting method is as follows: The cell sample is loaded by pneumatic drive, the thin-film beam splitter and the flat beam splitter are rotated to converge the microscopic imaging optical path and the Köhler coaxial illumination optical path into the Raman signal acquisition and detection optical path to observe the cell state in real time, and a periodic dielectric signal is applied to the dielectric focusing electrode. By controlling the dielectric on-off timing and adjusting the loading drive air pressure to control the flow rate of the cell sample, the cell sample passes through the dielectric focusing electrode for focusing, so as to realize that a single cell focuses and flows through and is captured at the outer tip of the end electrode in the dielectric focusing electrode, that is, the Raman excitation detection site; The Raman excitation optical path is turned on to emit a Raman excitation source, and the cell sample at the outer tip of the end electrode in the dielectric focusing electrode is focused by the visible light microscopic objective lens. The generated Raman signal enters the Raman signal acquisition and detection optical path, and by rotating the thin-film beam splitter and the flat beam splitter, it does not obstruct the Raman signal acquisition and detection optical path, so as to form a single-cell Raman spectrum to provide a criterion for sorting; Through the judgment and recognition of the host computer, the cells are sorted by applying a periodic dielectric signal to the dielectric sorting electrode; In the above process, the optical trap capture near-infrared laser optical path is kept closed, and the optothermal oscillation near-infrared laser optical path is intermittently turned on. The position of the optothermal oscillation near-infrared laser is adjusted by adjusting the optical path adjustment component, and a bubble oscillation force is generated through the optothermal effect to oscillate and separate the cells adhered to the outer tip of the end electrode in the dielectric focusing electrode; The optical trap capture flow Raman sorting method is as follows: Load the cell sample by pneumatic drive, rotate the thin film spectroscope and the flat spectroscope to converge the microscopic imaging optical path and the Köhler coaxial illumination optical path into the Raman signal acquisition and detection optical path to observe the cell state in real time. Introduce a double-layer sheath fluid into the chip to form a sheath flow, adjust the pneumatic drive pressure for loading to control the sample flow rate and the sheath fluid flow rate, and finally form a single-cell pipeline in the central flow field of the Raman excitation detection site in the chip for the cell sample; Turn on the optical trap to capture the near-infrared laser optical path, capture the cell sample at the Raman excitation detection site with the near-infrared laser captured by the optical trap, and at the same time pause the pneumatic drive pressure for loading; Turn on the Raman excitation optical path to emit a Raman excitation source, focus the cell sample at the outer tip of the end electrode in the dielectric focusing electrode through the visible light microscope objective lens. The generated Raman signal enters the Raman signal acquisition and detection optical path, and passes through the thin film spectroscope and the flat spectroscope without obstructing the Raman signal acquisition and detection optical path, thereby forming a single-cell Raman spectrum to provide a criterion for sorting; Judge and identify through the host computer, and sort the cell sample captured by the optical trap by moving the three-axis displacement stage.

10. The sorting method according to claim 9, characterized in that, The applicable cell sample size in the dielectric capture flow-through Raman sorting method is 5 - 60 μm; the applicable cell sample size in the optical trap capture flow-through Raman sorting method is less than 5 μm.

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