Flow system based on stimulated emission depletion ultra-thin optical sheet and micro-nanofluidic chip and its application

Abstract

The present invention proposes a flow system based on stimulated emission loss ultra-thin light sheet and micro-nanofluidic chip, including: a control module, a light source, a beam modulation module, a beam combining and focusing module, a calibration module, a micro-nanofluidic chip and a detection module. The present invention also discloses the application of the flow system based on stimulated emission loss ultra-thin light sheet and micro-nanofluidic chip to detect and analyze samples to be detected of biomedical and chemical materials. The flow system of the present invention adopts stimulated emission loss ultra-thin light sheet, breaks through the diffraction limit, improves the spatial resolution of the flow system, and can measure the number, size and posture of the samples to be detected. By using nanochannels, it can stretch the biomacromolecule samples, and measure and sequence the structure, material distribution and arrangement of the biomacromolecule samples. The flow system is matched with the synchronous control and scheduling of the control module, which can greatly improve the performance and function, efficiency, reliability and convenience.

Description

Technical Field

[0001] The invention belongs to the technical field of detection systems, and in particular relates to a flow system based on a stimulated emission loss ultra-thin optical sheet and a micro-nano fluidic chip. Background Art

[0002] Existing flow cytometers mainly detect and analyze biochemical samples through methods such as light spot excitation, impedance detection, and light sheet excitation. Among them, most flow cytometers use Gaussian light spots to excite samples to produce fluorescence for detection, and detect and analyze biochemical samples by collecting fluorescence signals to obtain the required information. Some use impedance electrical methods to measure the dielectric parameters of samples, and finally obtain the required information of biochemical samples. In addition, a small number of flow cytometers use light sheets to excite samples and detect fluorescence to obtain the required information of biochemical samples.

[0003] In the implementation of spot excitation, spatial resolution and detection range restrict each other. When the Gaussian spot area is very small, it is difficult to achieve large-scale measurement. Once the sample deviates from the detection area, it will be affected or cannot be detected. There are problems such as small detection area, low fault tolerance, and low reliability. When the Gaussian spot area is enlarged, it is difficult to measure the detailed information of the sample. Impedance-based detection based on electrical methods requires complex circuit design, and circuit-based detection methods are also easily affected by the electrical characteristics of the sample itself. Although there are many achievements in generating light sheets to excite samples such as cylindrical mirrors or spatial light modulators, the light sheet itself is still limited by the diffraction limit, resulting in low spatial resolution of the entire system and affected accuracy of the collected data. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a flow system based on stimulated emission loss ultra-thin light sheet and micro-nanofluidic chip in view of the above-mentioned deficiencies of the prior art. The system designs the stimulated emission loss ultra-thin light sheet for the first time, breaks through the diffraction limit, greatly improves the spatial resolution of the flow system, and can measure the number, size, and posture of the samples to be detected. By using nanochannels, it can also stretch the biomacromolecule samples, and measure and sequence the structure, material distribution, and arrangement of the biomacromolecule samples. The flow system is equipped with synchronous control and scheduling of the control module, which can greatly improve the performance and function, as well as efficiency, reliability, and convenience.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a flow system based on stimulated radiation loss ultra-thin optical sheet and micro-nanofluidic chip, characterized in that it includes: a control module, a light source, a beam modulation module, a beam combining and focusing module, a calibration module, a micro-nanofluidic chip and a detection module;

[0006] The control module is used to synchronously control and schedule the light source, beam modulation module, calibration module, and detection module, and collect the electrical signal of the sample to be detected obtained by the detection module, and then convert the electrical signal into digital data for transmission and storage, and process and analyze to obtain information of the sample to be detected;

[0007] There are two light sources, one emitting excitation light and the other emitting loss light under the control of the control module;

[0008] The beam modulation module is used to modulate the polarization and phase of the incident excitation light and loss light under the control of the control module to obtain the required excitation light sheet and loss light sheet;

[0009] The beam combining and focusing module is used to combine the excitation light sheet and the loss light sheet to generate a stimulated emission loss ultra-thin light sheet, and then focus it into the micro-nano channel on the micro-nano fluidic chip;

[0010] The calibration module is used to control the parameters of the beam modulation module in real time according to the strength of the light intensity signals of the excitation light sheet and the loss light sheet by using a filter mask under the control of the control module, so as to accurately and automatically align the excitation light sheet and the loss light sheet; the filter mask comprises two filters located on the optical path, the filter located in front of the optical path has a slit for calibrating the excitation light sheet, and the filter located behind the optical path has a slit for calibrating the loss light sheet;

[0011] The micro-nano fluidic chip is arranged on the detection module, and the micro-nano fluidic chip is divided into a micron channel chip or a nano channel chip. The micron channel chip has a micron channel, and the nano channel chip has a nano channel. The micron channel chip is used to drive the solution flow rate of the sample to be detected to be stably in the center position of the micron channel of the microfluidic chip under the entrainment of the sheath flow, so as to measure the number, size, posture and other information of the sample to be detected; the nano channel chip is used to drive the solution flow rate of the sample to be detected to be stably in the nano channel of the nano fluidic chip, so as to achieve the stretching of the biomacromolecule sample, and to achieve the measurement and sequencing of the structure, material distribution and arrangement of the biomacromolecule sample.

[0012] The detection module is used to align the positions of the stimulated radiation loss ultra-thin optical sheet and the micro-nano channel under the control of the control module, detect the fluorescence signal generated by the sample to be detected under the action of the stimulated radiation loss ultra-thin optical sheet and filter it, filter out the excitation light and loss light, and then receive it, convert the fluorescence signal with the excitation light and loss light filtered out into an electrical signal and transmit it to the control module.

[0013] Preferably, the control module includes an external high-speed data processing system and a computer; the computer contains an analysis algorithm, and the analysis algorithm is used to process and analyze the digital data converted by the control module into the electrical signal transmitted from the detection module to obtain information about the sample to be detected.

[0014] Preferably, the beam modulation module includes a quarter wave plate A, a half wave plate A, a lens A, a pinhole stop A, a lens B, a spatial light modulator A, a reflector A, a quarter wave plate B, a half wave plate B, a lens C, a pinhole stop B, a lens D, a spatial light modulator B, a reflector B, and a reflector C;

[0015] The quarter wave plate A, the half wave plate A, the lens A, the pinhole stop A, and the lens B are sequentially arranged on the optical path between the light source A and the spatial light modulator A, the spatial light modulator A is arranged on the optical path behind the lens B, and the spatial light modulator A is electrically connected to the control module;

[0016] The quarter wave plate B, the half wave plate B, the lens C, the pinhole stop B, and the lens D are sequentially arranged on the optical path between the light source B and the spatial light modulator B, the spatial light modulator B is arranged on the optical path behind the lens D, the reflector C is arranged on the reflected optical path of the reflector B, and the spatial light modulator B is electrically connected to the control module;

[0017] Preferably, the beam combining and focusing module comprises a dichroic mirror A, a dichroic mirror B and an objective lens A, wherein the dichroic mirror A is arranged on the reflection light path of the reflector A and the reflector C, the dichroic mirror B is parallel to the dichroic mirror A, and the objective lens A is arranged on the light path behind the dichroic mirror B;

[0018] Preferably, the calibration module includes an objective lens B, a reflector D, a filtering mask, a lens E, a multimode optical fiber A, a high-sensitivity optical detector A and a detector controller A; the detector controller A controls the high-sensitivity optical detector A, and the excitation light sheet and the loss light sheet emitted from the beam combining and focusing module pass through the objective lens B, the reflector D, the filtering mask, and the lens E in sequence, and are received by the multimode optical fiber A and then transmitted to the high-sensitivity optical detector A; the detector controller A includes an amplifier and a filter.

[0019] Preferably, the filtering mask is composed of two layers of vertical incidence filters with a special slit structure; the filter located in front of the optical path is provided with a slit a, slit a is highly transmittant to both excitation light and loss light, and the area outside slit a is highly reflective to excitation light and highly transmittant to loss light; two slits b are provided on the filter located behind the optical path, slit b is highly transmittant to both excitation light and loss light, and the area outside slit b is highly transmittant to excitation light and highly reflective to loss light.

[0020] Preferably, three liquid inlets and one liquid outlet are respectively provided on opposite sides of the micro-channel chip, and a micro-channel is provided between the liquid inlet and the liquid outlet in the middle, and the width of the micro-channel is at the micron level, and the width of the micro-channel does not exceed 10 μm; the two outer liquid inlets are connected to the micro-channel through a sheath flow channel, and the stimulated radiation loss ultra-thin optical sheet intersects the micro-channel perpendicularly, so as to measure the number, size, posture and other information of the samples to be detected flowing through the optical sheet;

[0021] The nanochannel chip is provided with a liquid inlet and a liquid outlet on opposite sides, respectively. The liquid inlet and the liquid outlet of the nanochannel chip are connected by a nanochannel. The width of the nanochannel is at the nanometer level and does not exceed 500nm. The stimulated radiation loss ultra-thin light sheet intersects the nanochannel vertically to achieve stretching of the biomacromolecule sample, and to achieve measurement and sequencing of the structure, material distribution and arrangement of the biomacromolecule sample.

[0022] Preferably, the detection module includes a controller of a translation stage, a translation stage, a micro-nano fluidic chip, a filter, a lens F, a multimode optical fiber B, a high-sensitivity optical detector B and a controller B of the detector; the controller of the translation stage controls the movement of the translation stage, and the micro-nano fluidic chip is mounted on the translation stage; the controller B of the detector controls the high-sensitivity optical detector B, the controller of the translation stage and the controller B of the detector are electrically connected to the control module, the micro-nano fluidic chip is arranged directly above the objective lens A, and the stimulated emission loss ultra-thin light sheet covers the micro-nano channel of the micro-nano fluidic chip, so that the sample to be detected is excited by the stimulated emission loss ultra-thin light sheet to generate a fluorescence signal, and the fluorescence signal is filtered out of the excitation light and the loss light in turn through the objective lens A, the dichroic mirror B, and the filter, and then focused by the lens F, received by the multi-mode optical fiber B, and then transmitted to the high-sensitivity optical detector B, the controller B of the detector and the control module in turn.

[0023] The present invention also discloses the above-mentioned flow system based on stimulated radiation loss ultra-thin optical sheet and micro-nanofluidic chip for detecting and analyzing samples to be detected of biomedical and chemical materials, and the specific steps include:

[0024] S1. Generate ultra-thin light sheet by stimulated emission loss: first, the control module controls light source A to generate excitation light and light source B to generate loss light, and then, under the control of the control module, the beam modulation module performs polarization and phase modulation on the excitation light and loss light to generate the required excitation light sheet and loss light sheet at a preset initial position; finally, the beam combining and focusing module completes the beam combining of the excitation light sheet and the loss light sheet to generate an ultra-thin light sheet by stimulated emission loss;

[0025] S2. Calibrate the positions of the excitation light sheet and the loss light sheet, including:

[0026] S201, the calibration module receives the light beams of the excitation light sheet and the loss light sheet through the objective lens B that is confocal with the objective lens A of the beam combining and focusing module, and allows them to pass through the filtering mask;

[0027] S202, turning on the excitation light and turning off the loss light through the control module, the calibration module detecting the intensity of the excitation light sheet passing through the filter mask, changing the position of the excitation light sheet through the beam modulation module under the control of the control module until the maximum excitation light sheet intensity is found, and determining that the excitation light sheet is aligned with the slit on the filter sheet of the filter mask located in front of the optical path;

[0028] S203, turning on the loss light and turning off the excitation light through the control module, the calibration module detecting the intensity of the loss light sheet passing through the filter mask, changing the position of the loss light sheet through the beam modulation module under the control of the control module until the maximum loss light sheet intensity is found, determining that the loss light sheet is aligned with the slit on the filter sheet of the filter mask located at the rear of the optical path, and then achieving accurate automatic alignment of the excitation light sheet and the loss light sheet;

[0029] S3, sample loading: according to the type of the sample to be detected, select a corresponding type of micro-nanofluidic chip and place it on the translation stage;

[0030] If a microchannel chip is selected, deionized water, ultrapure water or other solutions are input into the two outer liquid inlets of the microchannel to generate a sheath flow; then the solution of the sample to be detected is input into the liquid inlet in the middle of the microchannel to generate a sample flow;

[0031] If a nanochannel chip is selected, a solution of a sample to be detected is input into the liquid inlet of the nanochannel to generate a sample flow;

[0032] S4, high-precision detection: under the control of the control module, the detection module adjusts the position of the micro-nano fluidic chip to align the micro-nano channel with the stimulated radiation loss ultra-thin light sheet, ensuring that the stimulated radiation loss ultra-thin light sheet covers the entire micro-nano channel, and then the detection module filters the fluorescent signal generated by the sample to be detected, receives it after filtering out the excitation light and loss light, and converts it into an electrical signal. The control module collects the electrical signal and converts it into digital data for transmission and storage, and then processes and analyzes the digital data to obtain sample information. Due to the use of the stimulated radiation loss ultra-thin light sheet, the diffraction limit is broken, and a resolution of no more than 80nm can be achieved, thereby achieving high-precision detection.

[0033] Preferably, the processing and analyzing of the transmitted and stored digital data to obtain sample information specifically includes:

[0034] S401, the digital data is in the form of pulses, threshold filtering is performed on the pulses, and noise below the threshold is set to zero;

[0035] S402, identifying pulse intervals above the threshold, and ignoring pulse intervals whose widths do not meet the requirements, thereby finding rising edges and selecting pulse intervals;

[0036] S403, only the remaining selected pulse intervals are smoothed using a sliding average algorithm, a local maximum value and a position corresponding to the maximum value are found in the selected pulse intervals, and a falling edge and an end point of the falling edge are found in the selected pulse intervals according to the information of the maximum value;

[0037] S404, according to the falling edge of the selected pulse interval, combined with the rising edge, width and position of the selected pulse interval, check whether there are multiple pulses in the selected pulse interval, ignore the pulses that do not meet the requirements after the width test again, and finally determine the tested pulse interval and number;

[0038] S405, interpolating the pulse interval after the inspection to obtain enough data points for analysis, and on this basis calculating the half-height full width of the pulse interval after the inspection, which represents the sample size;

[0039] S406, looping the process of S402-S405, processing the next pulse intervals that have not been processed and analyzed in turn;

[0040] S407, statistics and analysis are performed on the data finally obtained, and finally relevant information of the sample is obtained.

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] 1. The flow system provided by the present invention uses the stimulated radiation loss ultra-thin optical sheet for the first time, which can increase the area of ​​the sample detection area during detection and improve the fault tolerance and reliability of the flow system;

[0043] 2. The flow system provided by the present invention adopts an optical detection method to avoid the problem that the electrical detection method is easily affected by the electrical characteristics of the sample itself, and can simplify the structure of the flow system compared to the electrical detection method;

[0044] 3. The stimulated emission loss ultra-thin light sheet used in the flow system provided by the present invention breaks through the diffraction limit, greatly improves the spatial resolution of the flow system, improves the detection accuracy of submicron / nanoscale samples, and can achieve the measurement of sample number, size, and posture. Especially when using a nanochannel chip, it can also achieve the stretching of biological macromolecule samples, and achieve the measurement and sequencing of the structure, material distribution and arrangement of biological macromolecule samples;

[0045] 4. The beam modulation module and calibration module of the flow system provided by the present invention can accurately and automatically align the positions of the excitation light sheet and the loss light sheet, greatly improving the quality and stability of the stimulated emission loss ultra-thin light sheet;

[0046] 5. The flow system provided by the present invention adopts a self-developed control module to synchronously control and schedule multiple light sources, beam modulation modules, calibration modules, and detection modules in the entire system, thereby realizing the generation and calibration of stimulated emission loss ultra-thin light sheets, and can also complete the detection and conversion of fluorescence signals, and finally complete the processing and analysis of data, so as to obtain the information required for samples in the fields of chemical and material analysis and biomedical testing in a one-stop, convenient and fast manner.

[0047] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of the structure of a flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip disclosed in Example 1 of the present invention.

[0049] Figure 2 It is a schematic diagram of the structure of the filtering mask disclosed in Embodiment 1 of the present invention.

[0050] Figure 3 This is a schematic diagram of the generation principle of the stimulated radiation loss ultra-thin optical sheet according to Example 1 of the present invention.

[0051] Figure 4 Schematic diagrams of the micro-nanofluidic chip of Example 1 of the present invention, wherein (a) is a schematic diagram of a micron channel chip, and (b) is a schematic diagram of a nano channel chip.

[0052] Figure 5 Schematic diagram of the cross-sectional detection area in the micro-nano channel of Example 1 of the present invention.

[0053] Figure 6 It is a schematic diagram of the application process of the present invention.

[0054] Description of reference numerals:

[0055] 1—light source A; 2—quarter wave plate A; 3—half wave plate A; 4—lens A; 5—pinhole stop A; 6—lens B; 7—spatial light modulator A; 8—reflector A; 9—light source B; 10—quarter wave plate B; 11—half wave plate B; 12—lens C; 13—pinhole stop B; 14—lens D; 15—spatial light modulator B; 16—reflector B; 17—reflector C; 18—dichroic mirror A; 19—dichroic mirror B; 20—objective lens A; 21—controller of translation stage; 22—translation stage; 23—microfluidic chip; 24—objective lens B; 25—reflector D; 26—filtering mask; 27—lens E; 28—multimode optical fiber A; 29—high-sensitivity optical detector A; 30—controller A of detector; 31—filter; 32—lens F; 33—multimode optical fiber B; 34—high-sensitivity optical detector B; 35—controller B of detector; 36—external high-speed data processing system; 37—computer. DETAILED DESCRIPTION

[0056] Example 1

[0057] like Figure 1 As shown, a flow system based on stimulated radiation loss ultra-thin optical sheet and micro-nano fluidic chip according to an embodiment of the present invention comprises: a control module, a light source, a beam modulation module, a beam combining and focusing module, a calibration module, a micro-nano fluidic chip, a detection module, and an analysis algorithm;

[0058] The control module is used to synchronously control and schedule the light source, beam modulation module, calibration module, and detection module, and to collect the electrical signal of the sample to be detected obtained by the detection module, and then convert the electrical signal into digital data for transmission and storage, and to process and analyze it to obtain information of the sample to be detected.

[0059] There are two light sources, one emitting excitation light and the other emitting loss light under the control of the control module;

[0060] The beam modulation module is used to modulate the polarization and phase of the incident excitation light and loss light under the control of the control module to obtain the required excitation light sheet and loss light sheet;

[0061] The beam combining and focusing module is used to combine the excitation light sheet and the loss light sheet to generate a stimulated emission loss ultra-thin light sheet, and then focus it into the micro-nano channel on the micro-nano fluidic chip;

[0062] The calibration module is used to combine the beam modulation module with the beam modulation module under the control of the control module, using a filter mask with a special slit structure (such as Figure 2As shown), the parameters of the beam modulation module are controlled in real time according to the strength of the light intensity signals of the excitation light sheet and the loss light sheet, and the excitation light sheet and the loss light sheet are accurately and automatically aligned; the filtering mask includes two filters located on the optical path, the filter located in front of the optical path has a slit for calibrating the excitation light sheet, and the filter located behind the optical path has a slit for calibrating the loss light sheet;

[0063] The micro-nano fluidic chip is arranged on the detection module. Figure 4 As shown, the micro-nanofluidic chip is divided into a micro-channel chip or a nano-channel chip, the micro-channel chip has a micro-channel, and the nano-channel chip has a nano-channel; the micro-channel chip is used to drive the solution flow rate of the sample to be detected to be stably in the center position of the micro-channel of the microfluidic chip under the entrainment of the sheath flow, so as to measure the number, size, posture and other information of the sample to be detected; the nano-channel chip is used to drive the solution flow rate of the sample to be detected to be stably in the nano-channel of the nano-fluidic chip, realize the stretching of the biomacromolecule sample, and realize the measurement and sequencing of the structure, material distribution and arrangement of the biomacromolecule sample;

[0064] The detection module is used to align the positions of the stimulated radiation loss ultra-thin optical sheet and the micro-nano channel under the control of the control module, detect the fluorescence signal generated by the sample to be detected under the action of the stimulated radiation loss ultra-thin optical sheet and filter it, filter out the excitation light and the loss light, and then receive it, convert the fluorescence signal from which the excitation light and the loss light are filtered out into an electrical signal and transmit it to the control module; the control module includes an analysis algorithm, and the analysis algorithm is used to process and analyze the digital data converted by the control module from the electrical signal transmitted by the detection module, so as to obtain information of the sample to be detected.

[0065] In this embodiment, the control module includes an external high-speed data processing system 36 and a computer 37. The computer 37 includes an analysis algorithm, which is used to process and analyze the digital data converted by the control module from the electrical signal transmitted by the detection module to obtain information about the sample to be detected. The light source includes a light source A1 and a light source B9, which are electrically connected to the computer 37 and controlled by the computer 37. The light source A1 generates excitation light under the control of the computer 37; the light source B9 generates loss light under the control of the computer 37.

[0066] In this embodiment, the beam modulation module includes a quarter wave plate A2, a half wave plate A3, a lens A4, a pinhole stop A5, a lens B6, a spatial light modulator A7, a reflector A8, a quarter wave plate B10, a half wave plate B11, a lens C12, a pinhole stop B13, a lens D14, a spatial light modulator B15, a reflector B16 and a reflector C17;

[0067] Specifically, the quarter wave plate A2, the half wave plate A3, the lens A4, the pinhole diaphragm A5, and the lens B6 are arranged in sequence on the optical path between the light source A1 and the spatial light modulator A7, the spatial light modulator A7 is arranged on the optical path behind the lens B6, the reflector A8 is arranged on the reflected optical path of the spatial light modulator A7, and the spatial light modulator A7 is electrically connected to the computer 37; more specifically, the light source A1 generates excitation light, and the excitation light passes through the quarter wave plate A2, the half wave plate A3, the lens A4, the pinhole diaphragm A5, and the lens A6 in sequence, and enters the spatial light modulator A7, the spatial light modulator A7 is electrically connected to the computer 37, and the computer 37 controls the spatial light modulator A7 to realize polarization and phase modulation of the excitation light to obtain the required excitation light sheet, and then the spatial light modulator A7 reflects the excitation light sheet, and then after being reflected by the reflector A8, it is incident on the dichroic mirror A18 to be combined with the loss light sheet.

[0068] The quarter wave plate B10, the half wave plate B11, the lens B12, the pinhole stop B13, and the lens B14 are arranged in sequence on the optical path between the light source B9 and the spatial light modulator B15, the spatial light modulator B15 is arranged on the optical path behind the lens D14, the reflector B16 is arranged on the reflected optical path of the spatial light modulator B15, and the spatial light modulator B15 is electrically connected to the computer 37; more specifically, the light source B9 generates loss light, which is sequentially transmitted through the quarter wave plate B10, the half wave plate B11, the lens B12, the pinhole stop B13, and the lens B14. B12, pinhole aperture B13, lens B14, incident spatial light modulator B15, spatial light modulator B15 is electrically connected to computer 37, computer 37 controls spatial light modulator B15 to realize polarization and phase modulation of loss light, and obtains the required loss light sheet, then spatial light modulator B15 reflects the loss light sheet, and then after being reflected by reflector B16, it is incident to reflector C17, and reflector C17 reflects the loss light sheet again, and it is incident to dichroic mirror A18, and is combined with the excitation light sheet to finally generate stimulated radiation loss ultra-thin light sheet.

[0069] The beam combining and focusing module includes a dichroic mirror A18, a dichroic mirror B19 and an objective lens A20. The dichroic mirror A18 is arranged on the reflection light path of the reflector A8 and the reflector C17. The dichroic mirror B19 is parallel to the dichroic mirror A18. The objective lens A20 is arranged on the light path behind the dichroic mirror B19.

[0070] In this embodiment, the calibration module includes an objective lens B24, a reflector D25, a filtering mask plate 26, a lens E27, a multimode optical fiber A28, a high-sensitivity optical detector A29 and a detector controller A30; the detector controller A29 includes an amplifier and a filter; the detector controller A30 controls the high-sensitivity optical detector A29, and the excitation light sheet and the loss light sheet emitted from the beam combining and focusing module pass through the objective lens B24, the reflector D25, the filtering mask plate 26, and the lens E27 in turn, and are received by the multimode optical fiber A28, and then transmitted to the high-sensitivity optical detector A29, processed by the detector controller A30, and transmitted to the external high-speed data processing system 36 for collection, conversion, storage, processing, and display, and finally transmitted to the computer 37 for analysis. The computer 37 controls the parameters of the beam modulation module according to the intensities of the received excitation light sheet and loss light sheet.

[0071] In this embodiment, the objective lens A20 is confocal with the objective lens B24 , and the objective lens B24 receives the light beams at the focal points of the excitation light sheet and the loss light sheet and reflects them onto the filtering mask 26 via the reflection mirror D25 .

[0072] like Figure 2 As shown, in this embodiment, the filtering mask is composed of two layers of vertical incidence filters with a special slit structure; the filter 26-1 located in front of the optical path is provided with a slit A, and the slit A is highly transmittant to both the excitation light and the loss light, and the area outside the slit A is highly reflective to the excitation light, and highly transmittant to the loss light; the filter 26-2 located behind the optical path is provided with two slits B, and the slit B is highly transmittant to both the excitation light and the loss light, and the area outside the slit B is highly transmittant to the excitation light, and highly reflective to the loss light.

[0073] like Figure 4 As shown in (a), three liquid inlets and one liquid outlet are respectively provided on opposite sides of the microfluidic chip, and a micron channel is provided between the liquid inlet and the liquid outlet in the middle, and the width of the micron channel is at the micron level, and the width of the micron channel does not exceed 10 μm; the other two liquid inlets are connected to the micron channel through a sheath flow channel, and the stimulated emission loss ultra-thin optical sheet intersects the micron channel perpendicularly, so as to measure the number, size, posture and other information of the samples to be detected flowing through the optical sheet;

[0074] like Figure 4 As shown in (b), a liquid inlet and a liquid outlet are respectively provided on opposite sides of the nanochannel chip, and the liquid inlet and the liquid outlet of the nanochannel chip are connected by a nanochannel, and the width of the nanochannel is at the nanometer level and does not exceed 500nm; the stimulated emission loss ultra-thin light sheet intersects the nanochannel perpendicularly to achieve stretching of the biomacromolecule sample, and to achieve measurement and sequencing of the structure, material distribution and arrangement of the biomacromolecule sample.

[0075] In this embodiment, the detection module includes a translation stage controller 21, a translation stage 22, a micro-nanofluidic chip 23, a filter 31, a lens F32, a multimode optical fiber B33, a high-sensitivity optical detector B34 and a detector controller B35; the translation stage controller 21 and the detector controller B35 are both electrically connected to the control module computer 37. The detector controller B35 includes an amplifier and a filter.

[0076] The micro-nano fluidic chip 23 is installed on the translation stage 22; the controller 21 of the translation stage controls the movement of the translation stage 22, and the controller B35 of the detector controls the high-sensitivity optical detector B34. The micro-nano fluidic chip 23 is arranged directly above the objective lens A20, and the stimulated radiation loss ultra-thin light sheet covers the micro-nano channel of the micro-nano fluidic chip 23, so that the sample to be detected is excited by the stimulated radiation loss ultra-thin light sheet to generate a fluorescence signal. The fluorescence signal is filtered out of the excitation light and the loss light in turn by the objective lens A20, the dichroic mirror B19, and the filter 31, and then focused by the lens F32, received by the multimode optical fiber B33, and then transmitted to the high-sensitivity optical detector B34 for detection, and the fluorescence signal with the excitation light and the loss light filtered out is converted into an electrical signal, which is transmitted to the external high-speed data processing system 36 through the detector controller B35 for collection, conversion, storage, processing, and display, and then transmitted to the computer 37 for processing and analysis to obtain relevant information of the sample to be detected.

[0077] In this embodiment, the high-sensitivity optical detector A29 and the high-sensitivity optical detector B34 are both photomultiplier tubes with a 10 5 -10 7 The gain can detect weak light. The translation stage is purchased commercially and has its own interface for connecting to the controller. The translation stage of model MAX311D produced by THORLABS can be used.

[0078] like Figure 6 As shown, the flow system based on stimulated emission depletion ultra-thin optical sheet and micro-nanofluidic chip is used to detect and analyze samples of biomedical and chemical materials to be detected. The specific steps include:

[0079] S1. Generating an ultra-thin optical sheet due to stimulated emission loss, specifically comprising:

[0080] S101, the computer 37 controls one light source to generate excitation light and another light source to generate loss light;

[0081] S102, the beam modulation module realizes polarization and phase modulation of the excitation light and the loss light under the control of the computer 37 of the control module, and generates the required excitation light sheet and loss light sheet at a preset initial position;

[0082] S103, the beam combining and focusing module completes the beam combining of the excitation light sheet and the loss light sheet to generate a stimulated emission loss ultra-thin light sheet;

[0083] S2. Calibration, including:

[0084] S201, receiving the light beams at the focal point of the excitation light sheet and the loss light sheet through the objective lens B24 located above the channel and having the same focal point as the objective lens A20 of the beam combining and focusing module, and irradiating the light beams onto the filtering mask 26;

[0085] S202, turn on the excitation light, turn off the loss light, and use the highly sensitive optical detector A29 to detect the intensity of the excitation light sheet passing through the filter mask 26. Under the control of the computer 37, the position of the excitation light sheet is changed through the beam modulation module until the maximum excitation light sheet intensity is found, and it is determined that the excitation light sheet is aligned with the slit on the filter sheet of the filter mask located in front of the optical path;

[0086] S203, turn on the loss light, turn off the excitation light, and use the highly sensitive optical detector A29 to detect the intensity of the loss light sheet passing through the filter mask 26. Under the control of the computer 37, the position of the loss light sheet is changed through the beam modulation module until the maximum loss light sheet intensity is found, and the loss light sheet is aligned with the slit on the filter sheet of the filter mask located behind the optical path, thereby achieving accurate automatic alignment of the excitation light sheet and the loss light sheet.

[0087] S3, sample loading, including:

[0088] S301, according to the type of sample to be detected, select a corresponding type of micro-nanofluidic chip and place it on the translation stage 23;

[0089] S302. If a microchannel chip is selected, a syringe pump or other device is used to connect the two outer liquid inlets to input deionized water, ultrapure water or other solutions to generate a sheath flow; then a syringe pump or other device is used to connect the middle liquid inlet to input the solution of the sample to be detected to generate a sample flow; if a nanochannel chip is selected, a syringe pump or other device is used to connect the liquid inlet to input the solution of the sample to be detected to generate a sample flow;

[0090] S4, high-precision detection: under the control of the control module, the translation stage 22 on the detection module adjusts the position of the micro-nano fluidic chip 23 to align the micro-nano channel with the stimulated emission loss ultra-thin optical sheet, ensuring that the stimulated emission loss ultra-thin optical sheet covers the entire micro-nano channel, such as Figure 5(b), the detection module then filters the fluorescent signal generated by the sample to be detected, receives it after filtering out the excitation light and loss light, and converts it into an electrical signal, the control module collects the electrical signal and converts it into digital data for storage, processing, and display, and then processes and analyzes the digital data to obtain sample information. The micro-nano channel is a micron channel or a nano channel;

[0091] In this example, the digital data is processed and analyzed to obtain sample information, including:

[0092] S401, the digital data is in the form of pulses, threshold filtering is performed on the pulses, and noise below the threshold (the threshold can be adjusted according to the sample) is set to zero;

[0093] S402, identifying pulse intervals above the threshold, and ignoring pulse intervals whose widths (the widths can also be adjusted according to the samples) do not meet the requirements, thereby finding the rising edge and selecting the pulse intervals;

[0094] S403, only the remaining selected pulse intervals are smoothed using a sliding average algorithm, a local maximum value and a position corresponding to the maximum value are found in the selected pulse intervals, and a falling edge and an end point of the falling edge are found in the selected pulse intervals according to the information of the maximum value;

[0095] S404, according to the falling edge of the selected pulse interval, combined with the rising edge, width and position of the selected pulse interval, check whether there are multiple pulses in the selected pulse interval, ignore the pulses that do not meet the requirements after the width test again, and finally determine the pulse interval and number after the test, which is the number of samples obtained;

[0096] S405, interpolate the pulse interval after the above test, obtain enough data points for analysis, and calculate the half-height full width of the pulse interval after the test. In actual analysis, for uniform structure samples, such as rectangular samples, the half-height full width can be used to obtain its posture; for spherical samples, the half-height full width can be used to obtain its diameter, i.e., size; for biological macromolecule samples, such as DNA molecules, the half-height full width can be used to obtain its structure, such as blood cells, the half-height full width can be used to obtain its material components; for mixed samples, the half-height full width can be used to obtain the sorting information of the mixed samples.

[0097] S406, looping the process of S402-S405, processing the next pulse intervals that have not been processed and analyzed in turn;

[0098] S407. Perform statistics and analysis on the data finally obtained, and finally obtain information such as the number, size, posture, structure, material distribution and arrangement of the samples.

[0099] When this embodiment is applied, for a micro-channel chip, a solution of 1 μm fluorescent particles can be used; for a nano-channel chip, a solution of 200 nm fluorescent particles can be used. The size of the fluorescent particles here is only for illustration and not for limitation.

[0100] The resolution of a microscope is disclosed in the prior art by the formula (Rayleigh criterion): d = 0.61λ / NA; d represents the resolution of the objective lens, in nm; λ represents the wavelength of the light source, in nm; NA represents the numerical aperture of the objective lens; that is, once the objective lens A20 of the system is selected, NA is fixed, and once the light sources A and B of the system are selected, the wavelength λ is also fixed, so once both the light source and the objective lens are selected, there is a theoretical resolution limit. In the prior art, a single light source is used for detection, such as a laser with a wavelength of λ = 488nm and an objective lens with NA = 1.4, that is, d = 0.61x488 / 1.4≈212.63nm. The resolution limit is preliminarily estimated to be above 200nm, which is the limitation of the diffraction limit and the theoretical limit of the prior art for sample fluorescence detection. The 200nm mark that cannot be broken through will be encountered, and samples smaller than 200nm are often unable to be detected. In fact, the traditional method cannot achieve such an ideal 200nm, and the resolution is often greater than 200nm.

[0101] Therefore, when only one beam of excitation light is used, the 200nm limit cannot be broken, and the excitation light will still excite a larger range, causing the samples in the larger range entering the channel to produce fluorescence. The principle can be understood as follows: Assuming that the excitation light forms a Gaussian spot in the micro-nano channel, Figure 5 As shown in (a), the diameter of the Gaussian spot is large, which is equivalent to the diffraction limit of 200nm mentioned above. When a sample smaller than 200nm (such as a 100nm sample) passes through the Gaussian spot, the detected data is still 200nm, which is inconsistent with the actual size. Therefore, the sample must be larger than 200nm to be detected. Moreover, the Gaussian spot may not completely cover the entire channel, resulting in the detection result being affected and becoming inaccurate or impossible to detect if the sample does not pass through the spot accurately.

[0102] However, if Figure 3As shown, the present invention adds a loss light sheet to an excitation light sheet, for example, using lasers with wavelengths λ=488nm and 592nm, so that the loss light sheet is sandwiched on both sides of the excitation light sheet, and the outer regions on both sides are de-excited, so that the outer regions on both sides generate stimulated radiation, and the wavelength of the stimulated radiation is different from the wavelength of the fluorescence generated by the excitation light sheet when the sample is excited. Then, the dichroic mirror B19 and the filter 31 can be used to filter out the stimulated radiation of the outer regions on both sides, compress the excitation range, and improve the spatial resolution. The present invention uses the stimulated radiation loss ultra-thin light sheet for the first time in the flow system, such as Figure 5 (b) The stimulated emission loss ultra-thin light sheet is thinner and can cover the entire channel, that is, the theoretical resolution is infinite, and the conservative estimate is no more than 80nm, which is a several-fold improvement in resolution. Due to such a high resolution, for biological macromolecule samples such as actin, neuronal synapses, and exosomes, whose typical size is far less than 200nm, traditional flow cytometers cannot detect them, but the flow system of the present invention can achieve detection.

[0103] At the same time, the present invention can obtain sample information conveniently and quickly in one stop, and has achieved a sampling rate of 500kHz. It is preliminarily estimated that it can reach 100k particles per second, while the current commercial flow system is about 35k particles per second. It will only be lower at the micro-nano scale, which is also an improvement of several times.

[0104] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip, It is characterized in that include: Control module, light source, beam modulation module, beam combining and focusing module, calibration module, micro-nanofluidic chip and detection module; The control module is used to synchronously control and schedule the light source, beam modulation module, calibration module, and detection module, and collect the electrical signal of the sample to be detected obtained by the detection module, and then convert the electrical signal into digital data for transmission and storage, and process and analyze to obtain information of the sample to be detected; There are two light sources, one emitting excitation light and the other emitting loss light under the control of the control module; The beam modulation module is used to modulate the polarization and phase of the incident excitation light and loss light under the control of the control module to obtain the required excitation light sheet and loss light sheet; The beam combining and focusing module is used to combine the excitation light sheet and the loss light sheet to generate a stimulated emission loss ultra-thin light sheet, and then focus it into the micro-nano channel on the micro-nano fluidic chip; The calibration module is used to control the parameters of the beam modulation module in real time according to the strength of the light intensity signals of the excitation light sheet and the loss light sheet by using a filter mask under the control of the control module, so as to accurately and automatically align the excitation light sheet and the loss light sheet; the filter mask comprises two filters located on the optical path, the filter located in front of the optical path has a slit for calibrating the excitation light sheet, and the filter located behind the optical path has a slit for calibrating the loss light sheet; The micro-nano fluidic chip is arranged on the detection module, and the micro-nano fluidic chip is divided into a micro-channel chip or a nano-channel chip. The micro-channel chip has a micro-channel, and the nano-channel chip has a nano-channel. The microchannel chip is used to drive the solution flow rate of the sample to be detected to be stably located in the center of the microchannel of the microfluidic chip under the sheath flow, so as to measure the number, size and posture information of the sample to be detected; the nanochannel chip is used to drive the solution flow rate of the sample to be detected to be stably located in the nanochannel of the nanofluidic chip, so as to achieve the stretching of the biomacromolecule sample, and to achieve the measurement and sequencing of the structure, material distribution and arrangement of the biomacromolecule sample; The detection module is used to align the positions of the stimulated radiation loss ultra-thin optical sheet and the micro-nano channel under the control of the control module, detect the fluorescence signal generated by the sample to be detected under the action of the stimulated radiation loss ultra-thin optical sheet and filter it, filter out the excitation light and loss light, and then receive it, convert the fluorescence signal with the excitation light and loss light filtered out into an electrical signal and transmit it to the control module.

2. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1, Features: The control module comprises an external high-speed data processing system (36) and a computer (37); the computer (37) comprises an analysis algorithm, and the analysis algorithm is used to process and analyze the digital data converted by the control module into the electrical signal transmitted by the detection module, so as to obtain information of the sample to be detected.

3. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1, Features: The beam modulation module comprises a quarter wave plate A (2), a half wave plate A (3), a lens A (4), a pinhole aperture A (5), a lens B (6), a spatial light modulator A (7), a reflector A (8), a quarter wave plate B (10), a half wave plate B (11), a lens C (12), a pinhole aperture B (13), a lens D (14), a spatial light modulator B (15), a reflector B (16), and a reflector C (17); The quarter wave plate A (2), the half wave plate A (3), the lens A (4), the pinhole aperture A (5), and the lens B (6) are sequentially arranged on the optical path between the light source A (1) and the spatial light modulator A (7); the spatial light modulator A (7) is arranged on the optical path behind the lens B (6); and the spatial light modulator A (7) is electrically connected to the control module; The quarter wave plate B (10), the half wave plate B (11), the lens C (12), the pinhole aperture B (13), and the lens D (14) are sequentially arranged on the optical path between the light source B (9) and the spatial light modulator B (15); the spatial light modulator B (15) is arranged on the optical path behind the lens D (14); the reflector C (17) is arranged on the reflected optical path of the reflector B (16); and the spatial light modulator B (15) is electrically connected to the control module.

4. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1, Features: The beam combining and focusing module comprises a dichroic mirror A (18), a dichroic mirror B (19) and an objective lens A (20), wherein the dichroic mirror A (18) is arranged on a reflection light path of a reflector A (8) and a reflector C (17), the dichroic mirror B (19) is parallel to the dichroic mirror A (18), and the objective lens A (20) is arranged on a light path behind the dichroic mirror B (19).

5. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1, Features: The calibration module comprises an objective lens B (24), a reflector D (25), a filter mask (26), a lens E (27), a multimode optical fiber A (28), a high-sensitivity optical detector A (29) and a detector controller A (30); the detector controller A (30) controls the high-sensitivity optical detector A (29), and the excitation light sheet and the loss light sheet emitted from the beam combining and focusing module pass through the objective lens B (24), the reflector D (25), the filter mask (26) and the lens E (27) in sequence, are received by the multimode optical fiber A (28), and are then transmitted to the high-sensitivity optical detector A (29); the detector controller A (30) comprises an amplifier and a filter.

6. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1 or 5, Features: The filtering mask (26) is composed of two layers of vertically incident filters with a special slit structure; the filter located in front of the optical path is provided with a slit a, and the slit a is highly transmittant to both the excitation light and the loss light, and the area outside the slit a is highly reflective to the excitation light and highly transmittant to the loss light; the filter located behind the optical path is provided with two slits b, and the slit b is highly transmittant to both the excitation light and the loss light, and the area outside the slit b is highly transmittant to the excitation light and highly reflective to the loss light.

7. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1, Features: The microchannel chip is provided with three liquid inlets and one liquid outlet on opposite sides, and a microchannel is provided between the liquid inlet and the liquid outlet in the middle, wherein the width of the microchannel is at the micron level and does not exceed 10 μm; The two outer liquid inlets are connected to the micron channel through the sheath flow channel, and the stimulated emission loss ultra-thin light sheet intersects the micron channel vertically, so as to measure the number, size and posture information of the samples to be detected flowing through the light sheet; The nanochannel chip is provided with a liquid inlet and a liquid outlet on opposite sides, respectively. The liquid inlet and the liquid outlet of the nanochannel chip are connected by a nanochannel. The width of the nanochannel is at the nanometer level and does not exceed 500nm. The stimulated radiation loss ultra-thin light sheet intersects the nanochannel vertically to achieve stretching of the biomacromolecule sample, and to achieve measurement and sequencing of the structure, material distribution and arrangement of the biomacromolecule sample.

8. The flow system based on stimulated emission loss ultra-thin optical sheet and micro-nanofluidic chip according to claim 1 or 4, Features: The detection module comprises a translation stage controller (21), a translation stage (22), a micro-nanofluidic chip (23), a filter (31), a lens F (32), a multimode optical fiber B (33), a high-sensitivity optical detector B (34) and a detector controller B (35); the translation stage controller (21) controls the movement of the translation stage (22), and the micro-nanofluidic chip (23) is mounted on the translation stage (22); the detector controller B (35) controls the high-sensitivity optical detector B (34), and the translation stage controller (21) and the detector controller B (35) are both connected to the controller The control module is electrically connected, the micro-nanofluidic chip (23) is arranged directly above the objective lens A (20), and the stimulated emission loss ultra-thin optical sheet covers the micro-nano channel of the micro-nanofluidic chip (23), so that the sample to be detected is excited by the stimulated emission loss ultra-thin optical sheet to generate a fluorescence signal, and the fluorescence signal is filtered out of the excitation light and the loss light in sequence through the objective lens A (20), the dichroic mirror B (19), and the filter (31), and then focused by the lens F (32), received by the multimode optical fiber B (33), and then transmitted to the high-sensitivity optical detector B (34), the controller B (35) of the detector, and the control module in sequence.

9. Application of a streaming system, Features: The flow system based on stimulated emission depletion ultra-thin optical sheet and micro-nanofluidic chip as described in any one of claims 1 to 8 is used to detect and analyze samples to be detected of biomedical and chemical materials, and the specific steps include: S1. Generate a stimulated emission loss ultra-thin light sheet: first, the control module controls the light source to generate the excitation light, and the other light source to generate the loss light. Then, under the control of the control module, the beam modulation module performs polarization and phase modulation on the excitation light and the loss light to generate the required excitation light sheet and loss light sheet at a preset initial position. Finally, the beam combining and focusing module completes the beam combining of the excitation light sheet and the loss light sheet to generate a stimulated emission loss ultra-thin light sheet. S2. Calibrate the positions of the excitation light sheet and the loss light sheet, including: S201, the calibration module receives the light beams from the excitation light sheet and the loss light sheet, and allows them to pass through the filtering mask (26); S202, turning on the excitation light and turning off the loss light through the control module, the calibration module detecting the intensity of the excitation light sheet passing through the filter mask (26), changing the position of the excitation light sheet through the beam modulation module under the control of the control module until the maximum excitation light sheet intensity is found, and determining that the excitation light sheet is aligned with the slit on the filter sheet of the filter mask located in front of the optical path; S203, turning on the loss light and turning off the excitation light through the control module, the calibration module detecting the intensity of the loss light sheet passing through the filter mask (26), changing the position of the loss light sheet through the beam modulation module under the control of the control module until the maximum loss light sheet intensity is found, determining that the loss light sheet is aligned with the slit on the filter sheet of the filter mask located behind the optical path, and thus achieving accurate automatic alignment of the excitation light sheet and the loss light sheet; S3, sample loading: according to the type of sample to be detected, select a corresponding type of micro-nanofluidic chip (23) and place it on the detection module; If a micro-channel chip is selected, deionized water and ultrapure water are input into the two liquid inlets on the outside of the micro-channel to generate a sheath flow; then a solution of a sample to be detected is input into the liquid inlet in the middle of the micro-channel to generate a sample flow; If a nanochannel chip is selected, a solution of a sample to be detected is input into the liquid inlet of the nanochannel to generate a sample flow; S4, high-precision detection: under the control of the control module, the detection module adjusts the position of the micro-nano fluidic chip (23) so that the micro-nano channel is aligned with the stimulated radiation loss ultra-thin light sheet, ensuring that the stimulated radiation loss ultra-thin light sheet covers the entire micro-nano channel. Then, the detection module filters the fluorescence signal generated by the sample to be detected, receives it after filtering out the excitation light and loss light, and converts it into an electrical signal. The control module collects the electrical signal and converts it into digital data for transmission and storage, and then processes and analyzes the digital data to obtain sample information.

10. Application of a streaming system according to claim 9, Features: The processing and analysis of the transmitted and stored digital data to obtain sample information specifically includes: S401, the digital data is in the form of pulses, threshold filtering is performed on the pulses, and noise below the threshold is set to zero; S402, identifying pulse intervals above the threshold, and ignoring pulse intervals whose widths do not meet the requirements, thereby finding rising edges and selecting pulse intervals; S403, only the remaining selected pulse intervals are smoothed using a sliding average algorithm, a local maximum value and a position corresponding to the maximum value are found in the selected pulse intervals, and a falling edge and an end point of the falling edge are found in the selected pulse intervals according to the information of the maximum value; S404, according to the falling edge of the selected pulse interval, combined with the rising edge, width and position of the selected pulse interval, check whether there are multiple pulses in the selected pulse interval, ignore the pulses that do not meet the requirements after the width test again, and finally determine the tested pulse interval and number; S405, interpolating the pulse interval after the inspection to obtain enough data points for analysis, and on this basis calculating the full width at half maximum of the pulse interval after the inspection; S406, looping the process of S402-S405, processing the next pulse intervals that have not been processed and analyzed in turn; S407, statistics and analysis are performed on the data finally obtained, and finally relevant information of the sample is obtained.

Images