A droplet microfluidics-based label-free live cell screening system and method

Abstract

This invention provides a label-free live cell screening system and method based on droplet microfluidics. The system includes: a microfluidic chip with fluorescence signal monitoring sites, imaging sites, and sorting sites; a fluorescence detection unit configured to excite and acquire fluorescence signals at the fluorescence signal monitoring sites; an imaging unit configured to acquire images of droplets at the imaging sites; a sorting unit configured to sort droplets encapsulating target cells at the sorting sites; and a signal processor that, when a droplet is detected passing through the fluorescence signal monitoring site based on the fluorescence signal, triggers the imaging unit to acquire images of the droplet at the imaging site; and when the signal processor determines, based on image analysis, that the droplet encapsulates a target cell, triggers the sorting unit to sort the droplet encapsulating the target cell at the sorting site. This invention utilizes fluorescence signals to monitor droplets, improving monitoring efficiency and accuracy, thereby significantly increasing the precision and throughput of droplet sorting.

Description

Technical Field

[0001] This invention relates to the field of cell sorting technology, specifically to a label-free live cell screening system and method based on droplet microfluidics. Background Technology

[0002] Label-free live cell screening refers to isolating target cells from a population without any pretreatment, based solely on the cells' physiological phenotypic characteristics while preserving cell viability, for subsequent culture, analysis, and utilization. Label-free live cell screening has a wide range of applications in the field of biology, particularly in screening microbial cells with significant industrial or medical value. Droplet microfluidics is considered one of the best technical approaches for achieving label-free live cell screening. This is mainly because a large number of microdroplets can provide each cell in the population with an independent growth space. Cells grow and exhibit their physiological phenotypes within their respective spaces. Large or small molecules secreted by cells are confined within their individual droplet spaces, completely eliminating interference between cells and making it easier to identify and screen cells with specific phenotypic characteristics.

[0003] Currently, label-free live cell screening methods based on droplet microfluidics can be categorized into five types according to their cell phenotypic detection principles: optical absorption / scattering, Raman spectroscopy, mass spectrometry, electrochemical methods, and imaging methods. Among these, Raman spectroscopy, mass spectrometry, and electrochemical methods can only detect cellular metabolites, thus limiting their applications; optical absorption / scattering methods can only detect changes in cell number, also limiting their applications significantly; imaging methods can detect various physiological phenotypic characteristics, including cell morphology, number, and metabolic activity, and have a broader range of applications. Therefore, developing imaging-based droplet microfluidic label-free live cell screening technology is of great significance.

[0004] Imaging-based droplet microfluidic label-free live cell screening methods typically include the following steps:

[0005] (1) Use microfluidic chips to generate microdroplets of uniform size, and encapsulate single or multiple cells into the microdroplets at the same time as generating the microdroplets;

[0006] (2) Collect and incubate microdroplets to allow cells within the microdroplets to grow and exhibit physiological phenotypes;

[0007] (3) Inject microdroplets into a microfluidic droplet sorting chip, acquire images of each microdroplet, and identify target cells by analyzing the physiological phenotypic characteristics of cells within the microdroplets;

[0008] (4) After the target cells are identified, the microdroplets containing the target cells are sorted out, and the target cells are obtained by recycling the sorted microdroplets, thus achieving the purpose of cell screening.

[0009] The core of the above method lies in the microdroplet image acquisition and cell physiological phenotypic feature analysis in step (3). The acquisition and analysis speed of microdroplet image data directly determines the throughput of cell screening; the quality of the microdroplet images and the performance of the cell physiological phenotypic feature analysis directly affect the sensitivity and accuracy of cell screening. Currently, a series of methods for microdroplet image acquisition and cell physiological phenotypic feature analysis have been proposed. These methods can be divided into two categories based on the image acquisition method:

[0010] I. Continuous Image Acquisition Method: A high-speed camera is used to continuously acquire images of the microfluidic chip channel at a fixed frequency, and the presence of flowing microdroplets in each acquired image is analyzed in real time. When microdroplets are detected in the acquired image, the physiological phenotypic characteristics of the cells within the droplets are further analyzed, and it is determined whether the cells within the microdroplets are target cells. If the cells are determined to be target cells, the droplet sorting function is executed to recover the target cells.

[0011] In continuous image acquisition methods, most of the images captured by high-speed cameras are invalid data, i.e., images of droplets that were not captured. However, to find images of captured droplets, this invalid data still needs to be analyzed one by one. Image analysis is the rate-limiting step of the entire technique, and the analysis of a large amount of invalid image data severely affects the efficiency of droplet sorting and limits the cell screening throughput. Furthermore, the analysis of invalid data wastes significant hardware resources, resulting in higher equipment costs to achieve the same cell screening effect. This method can achieve a droplet sorting frequency of 10-30Hz.

[0012] II. Triggered Image Acquisition Method: A photodetector is used to monitor microdroplets flowing through the microfluidic chip channel. When a droplet passes through the chip channel, the photodetector detects a pulsed change in brightness, triggering a high-speed camera to acquire an image of the microdroplet. The image is then analyzed in real time to identify the droplet outline, analyze the physiological phenotypic characteristics of the cells within the droplet, and determine whether the cells within the microdroplet are target cells. Once a target cell is identified, a droplet sorting function is executed to recover the target cell.

[0013] The hardware configuration of the triggered image acquisition method is as follows: a microfluidic chip is placed on the stage of an inverted microscope, and a high-speed camera is mounted on the imaging optical path of the inverted microscope, using transmitted light illumination to generate bright-field microscopic images. A beam splitter divides the light signal acquired by the objective lens into two parts; one part is transmitted to the high-speed camera for imaging, and the other part is transmitted to a photodetector for droplet monitoring. Irregular pulse signals caused by the passage of droplets are converted into pulse level signals and transmitted to the high-speed camera to trigger image acquisition of the droplets. The output of the high-speed camera is connected to a computer host, and the image data is transmitted to the computer host for analysis. When a target cell is detected and droplet sorting is required, the computer host sends a sorting command to a device capable of generating sorting electrical pulse signals. Finally, the signal is transmitted to the microfluidic droplet sorting chip to perform droplet sorting. The droplet sorting frequency of this method can reach 40-100Hz.

[0014] Existing triggered image acquisition methods monitor microdroplets flowing through the channels of microfluidic chips by detecting changes in transmitted light intensity. This method acquires images only after droplet detection, avoiding the collection of large amounts of invalid image data. However, it also introduces new problems, mainly including:

[0015] Microdroplets are extremely small, and the changes in transmitted light intensity caused by them are very weak. Therefore, the signal-to-noise ratio of microdroplet monitoring signals based on this principle is very low, resulting in low sensitivity and accuracy, and consequently, low droplet screening throughput. To accurately detect droplets, a pinhole is needed to narrow the detection range to a size close to the droplet diameter. However, selecting the pinhole size and setting its position relative to the high-speed camera's field of view are very complex, and post-calibration is also extremely cumbersome.

[0016] High-speed camera imaging and photodetector monitoring of microdroplets use the same illumination source. To simultaneously meet the requirements of imaging and droplet monitoring, the illumination intensity, camera exposure time, and photodetector sensitivity need to be adjusted to achieve a high degree of matching, which greatly increases the workload of setting up each device. Furthermore, to ensure the stability of the microdroplet monitoring signal, the intensity of the illumination source needs to be fixed during the detection process. Therefore, the illumination intensity for imaging cannot be arbitrarily adjusted according to different imaging needs, which greatly reduces the flexibility of this method.

[0017] Photodetectors use transmitted light to monitor microdroplet signals, so imaging of microdroplets and their internal cells can only be done using bright-field imaging based on transmitted light illumination, which greatly limits the application scope of this method.

[0018] Microdroplet monitoring based on transmitted light intensity is susceptible to various factors. For example, the composition of substances such as cells within the microdroplet can cause variations in light intensity, and different refractive indices at the microdroplet interface formed by using different oil-water systems can lead to changes in the rate of change of the light signal. These issues can reduce the sensitivity and accuracy of microdroplet monitoring, thereby affecting the reliability of cell screening. Summary of the Invention

[0019] To address the shortcomings of existing technologies, this invention provides a label-free live cell screening system and method based on droplet microfluidics, aiming to solve the problem of how to improve the sensitivity and accuracy of droplet monitoring and thus increase screening throughput.

[0020] To address the aforementioned problems, one aspect of the present invention is to provide a label-free live cell screening system based on droplet microfluidics, the system comprising:

[0021] A microfluidic chip includes a microfluidic channel configured for transporting droplets; a fluorescence signal monitoring site, an imaging site, and a sorting site are sequentially arranged on the microfluidic channel.

[0022] A fluorescence detection unit is configured to emit excitation light toward the fluorescence signal monitoring site to excite a fluorescence signal and to acquire the fluorescence signal;

[0023] An imaging unit is configured to acquire an image of the droplet at the imaging site;

[0024] The sorting unit is configured to sort droplets containing target cells at the sorting site;

[0025] A signal processor is connected to the fluorescence detection unit, the imaging unit, and the sorting unit, respectively.

[0026] Specifically, when the signal processor detects a droplet passing through the fluorescence signal monitoring site based on the fluorescence signal, it triggers the imaging unit to acquire an image of the droplet at the imaging site; when the signal processor determines that the droplet contains a target cell based on the image, it triggers the sorting unit to sort out the droplet containing the target cell at the sorting site.

[0027] In the specific scheme, the microfluidic channel includes a main delivery channel, a first injection channel and a second injection channel connected to a first end of the main delivery channel, and a first output channel and a second output channel connected to a second end of the main delivery channel; the fluorescence signal monitoring site, imaging site and sorting site are arranged sequentially on the main delivery channel from the first end to the second end, and the sorting site is arranged near the intersection of the first output channel and the second output channel.

[0028] In the specific embodiment, the microfluidic chip is connected to a fluid driving device, which is used to inject droplets into the first injection channel and inject a continuous liquid phase into the second injection channel, and drive the droplets to flow from the first end of the main delivery channel toward the second end.

[0029] In the specific scheme, the fluorescence detection unit includes an excitation light module and a detection module. The excitation light module includes a light source and a beam shaping element, and the detection module includes a converging lens and a high-speed detector. The excitation light emitted by the light source passes through the beam shaping element and is incident on the fluorescence signal monitoring site to excite fluorescence. The excited fluorescence is converged by the converging lens and incident on the high-speed detector. The high-speed detector sends the collected fluorescence signal to the signal processor.

[0030] In a specific embodiment, the imaging unit includes an illumination module, an objective lens, an imaging lens, and a high-speed imaging camera. The illumination module and the objective lens are located on opposite upper and lower sides of the microfluidic channel. The illumination light emitted by the illumination module is collected by the objective lens and focused onto the high-speed imaging camera via the imaging lens. The high-speed imaging camera then sends the acquired image signal to the signal processor.

[0031] In the specific scheme, the sorting unit includes a voltage amplifier and an electrode. The electrode is disposed in the microfluidic chip and is located on the side of the main delivery channel and near the sorting site. Under the control of the signal processor, the voltage amplifier applies a voltage to the electrode to generate a non-uniform electric field, thereby sorting out the droplets encapsulating the target cells.

[0032] Another aspect of the present invention is to provide a label-free live cell screening method based on droplet microfluidics, employing the label-free live cell screening system described above, the method comprising the steps of:

[0033] The driving droplet flows from the first end to the second end in the microfluidic channel of the microfluidic chip along with the continuous liquid phase;

[0034] The fluorescence detection unit is controlled to emit excitation light toward the fluorescence signal monitoring site to excite the fluorescence signal of the droplet, and the fluorescence signal is collected and fed back to the signal processor;

[0035] The signal processor determines whether a droplet has passed through the fluorescence signal monitoring site based on the fluorescence signal; if so, it triggers the imaging unit to acquire an image of the droplet at the imaging site and feeds it back to the signal processor.

[0036] The signal processor determines whether the droplet contains a target cell based on the image; if so, it triggers the sorting unit to sort the droplet containing the target cell at the sorting point.

[0037] In a specific scheme, determining whether a droplet has passed through the fluorescence signal monitoring site includes: the signal processor continuously receives a fluorescence signal sequence fed back from the fluorescence detection unit, and compares the fluorescence signal sequence with a set fluorescence signal threshold: when the signal value of the fluorescence signal sequence first jumps from below the threshold to above the threshold and generates a rising edge, and then jumps from above the threshold to below the threshold and generates a falling edge, it is determined that a droplet has passed through the fluorescence signal monitoring site.

[0038] In a specific scheme, determining whether the droplet encapsulates the target cell based on the image includes: performing background filtering on the original image of the acquired droplet; applying morphological operators to process the filtered image to identify the droplet boundary and cells within the droplet, obtaining mask morphology data of the cells; calculating and obtaining the phenotypic features of the mask-marked cells within the droplet and comparing them with preset conditions; if the phenotypic features meet the preset conditions, it is determined that the droplet encapsulates the target cell.

[0039] In a specific scheme, triggering the sorting unit to sort the droplets encapsulating target cells at the sorting point includes: the signal processor controlling the sorting unit to generate a non-uniform electric field at the sorting point, causing the flow path of the droplets encapsulating target cells to deviate, and controlling the droplets encapsulating target cells to flow through a predetermined output channel, thereby screening out the droplets encapsulating target cells.

[0040] The label-free live cell screening system and method based on droplet microfluidics provided in this invention uses excitation light to excite the fluorescence signal inherent in the droplets, and identifies and determines the droplets based on the fluorescence signal, which has the following beneficial effects:

[0041] (1) No need to use pinholes to limit the monitoring area, making hardware setup simpler; the excitation light will form a light spot in the imaging field of the high-speed camera. As long as the microfluidic chip channel is aligned with the light spot, the monitoring of the microdroplets flowing through the channel can be realized. The device setup is more convenient and does not require frequent calibration.

[0042] (2) The monitoring of droplets and high-speed camera imaging use light signals of different wavelengths that do not interfere with each other. The intensity of the illumination source used for imaging can be adjusted at will as needed. Furthermore, the imaging modes of cells inside droplets are more diverse. In addition to bright field imaging, dark field, phase contrast, DIC and other imaging modes can also be used, greatly expanding the application range.

[0043] (3) The signal-to-noise ratio of microdroplet monitoring signals is higher, and the signal is not affected by the contents of microdroplets, nor by the physical and chemical properties of microdroplets themselves. Therefore, the monitoring signal is more stable, thereby improving the sensitivity and accuracy of droplet monitoring, improving the reliability of cell screening, and also increasing the cell screening throughput. The cell droplet sorting frequency can reach more than 1000 Hz. Attached Figure Description

[0044] Figure 1 This is a structural block diagram of the label-free live cell screening system provided in an embodiment of the present invention;

[0045] Figure 2 This is a schematic diagram of the microfluidic channel in an embodiment of the present invention. Detailed Implementation

[0046] To achieve the foregoing and related objectives, the present invention includes the features fully described below and particularly pointed out in the claims. Certain illustrative aspects and embodiments of the invention are set forth in detail in the following description and drawings. However, these are merely indications of a few of the various ways in which the principles of the invention can be employed. Other objects, advantages, and novel features of the invention will become apparent when considered in conjunction with the accompanying drawings and the following detailed description based on the invention.

[0047] It should also be noted that, in order to avoid obscuring the invention with unnecessary details, only the structures and / or processing steps closely related to the solution according to the invention are shown in the accompanying drawings, while other details that are not closely related to the invention are omitted.

[0048] To address the low throughput issues in existing imaging-based droplet microfluidic label-free live cell screening methods, and the numerous problems arising from the use of the same illumination source for both the droplet photodetector and the imaging device for acquiring droplet images in triggered image acquisition methods, this invention provides a droplet microfluidic label-free live cell screening system and method. This system uses excitation light to excite the fluorescence signal inherent in the droplet, identifies and determines the droplet based on the fluorescence signal, and then triggers the imaging device to take a picture based on the determination result. This improves monitoring efficiency and accuracy. Combined with droplet image analysis, it significantly enhances the precision and throughput of droplet sorting, enabling high-throughput screening of different phenotypic cell populations within droplets.

[0049] Based on the above concept, the present invention first provides a label-free live cell screening system based on droplet microfluidics, the system including a microfluidic chip, a fluorescence detection unit, an imaging unit, a sorting unit and a signal processor.

[0050] The microfluidic chip includes at least a microfluidic channel configured for transporting droplets; and a fluorescence signal monitoring site, an imaging site, and a sorting site are sequentially arranged on the microfluidic channel. The fluorescence detection unit is configured to emit excitation light to the fluorescence signal monitoring site to excite a fluorescence signal and to acquire the fluorescence signal. The imaging unit is configured to acquire an image signal of the droplet at the imaging site. The sorting unit is configured to sort droplets encapsulating target cells at the sorting site; the signal processor is connected to the fluorescence detection unit, the imaging unit, and the sorting unit respectively.

[0051] The signal processor identifies and determines whether a droplet passes through the fluorescence signal monitoring site based on the fluorescence signal. If so, it triggers the imaging unit to acquire an image of the droplet at the imaging site. The signal processor also determines whether the droplet encapsulates a target cell based on the image. If so, it triggers the sorting unit to sort the droplet encapsulating the target cell at the sorting site.

[0052] In this embodiment, refer to Figure 1 and Figure 2 The microfluidic chip 1 is disposed on the stage 2. The microfluidic chip 1 includes a microfluidic channel 10, comprising a main delivery channel 11, a first injection channel 12 and a second injection channel 13 connected to a first end of the main delivery channel 11, and a first output channel 14 and a second output channel 15 connected to a second end of the main delivery channel 11. The first injection channel 12 is used to inject droplets, and the second injection channel 13 is used to inject a continuous liquid phase. The first output channel 14 is used for the outflow of sorted droplets encapsulating target cells, and the second output channel 15 is used for the outflow of other droplets besides those encapsulating target cells, as well as other fluids. It should be noted that the droplets injected through the first injection channel 12 include droplets encapsulating target cells, droplets encapsulating non-target cells, and blank droplets without any cells encapsulated.

[0053] Furthermore, such as Figure 2 As shown, fluorescence signal monitoring sites 16, imaging sites 17, and sorting sites 18 are sequentially arranged on the main delivery channel 11. The fluorescence signal monitoring sites 16, imaging sites 17, and sorting sites 18 are arranged sequentially on the main delivery channel 11 in the direction from the first end to the second end (i.e., the flow direction of the droplets), and the sorting sites 18 are arranged near the intersection of the first output channel 14 and the second output channel 15.

[0054] Furthermore, such as Figure 1As shown, the microfluidic chip 1 is connected to a fluid driving device 3, which is used to inject droplets into the first injection channel 12 and inject a continuous liquid phase into the second injection channel 13, and drive the droplets to flow from the first end of the main delivery channel 11 toward the second end.

[0055] In this embodiment, the fluorescence detection unit includes an excitation light module and a detection module. For example... Figure 1 As shown, the excitation light module includes a light source 31 and a beam shaping element 32, and the detection module includes a converging lens 34 and a high-speed detector 35. The excitation light emitted from the light source 31 passes through the beam shaping element 32 and is incident on the fluorescence signal monitoring site 16 to excite fluorescence. The excited fluorescence is then converged by the converging lens 34 and incident on the high-speed detector 35. The high-speed detector 35 is connected to the signal processor 4, and the high-speed detector 35 sends the collected fluorescence signal S1 to the signal processor 4.

[0056] The light source 31 is provided by a laser or a light-emitting diode (LED) light source; in this embodiment, a laser is selected, meaning the excitation light is laser light. The beam shaping element 32 is used to shape the excitation light emitted from the light source 31 into a specific beam. The beam shaping element 32 can be selected from a collimator for collimating the beam into parallel light or a cylindrical lens group for shaping a circular light spot into a linear light spot; in this embodiment, a cylindrical lens group is selected to improve the signal-to-noise ratio of signal detection. In other embodiments, the beam shaping element 32 is not limited to the collimator and cylindrical lens group mentioned above.

[0057] Furthermore, in this embodiment, as Figure 1 As shown, since the optical path of the excitation light overlaps with the optical path of the fluorescence signal, the fluorescence detection unit is also provided with a first beam splitter 33. The excitation light emitted by the light source 31 passes through the beam shaping element 32 and is reflected by the first beam splitter 33 before being incident on the fluorescence signal monitoring site 16 to excite fluorescence. The excited fluorescence is transmitted through the first beam splitter 33 and then converged by the converging lens 34 to be incident on the high-speed detector 35.

[0058] Based on the fluorescence detection unit described in the above embodiments, the excitation light module continuously emits excitation light to the fluorescence signal monitoring site 16 to continuously excite fluorescence, while the detection module continuously collects fluorescence signals. When a droplet passes through the fluorescence signal monitoring site 16 in the main transport channel 11 along with a continuous liquid phase, the fluorescence signal excited from the droplet differs from the fluorescence signal excited from other fluids. Therefore, the signal processor 4 can identify whether the current object passing through the fluorescence signal monitoring site 16 is a droplet based on the received fluorescence signal, thereby monitoring the droplet and determining whether to trigger the imaging unit to capture images based on the monitoring results.

[0059] In this embodiment, as Figure 1 As shown, the imaging unit includes an illumination module 21, an objective lens 22, an imaging lens 24, and a high-speed imaging camera 25. The illumination module 21 and the objective lens 22 are located on opposite sides of the microfluidic chip 1, specifically on opposite sides of the microfluidic channel 10. Figure 1 In this configuration, the illumination module 21 is located above the microfluidic chip 1, while the objective lens 22 is located below the microfluidic chip 1.

[0060] Specifically, the illumination light emitted by the illumination module 21 is collected by the objective lens 22 and focused onto the high-speed imaging camera 25 via the imaging lens 24. The high-speed imaging camera 25 is connected to the signal processor 4, which controls the high-speed imaging camera 25 to acquire images, and the high-speed imaging camera 25 sends the acquired image signal S2 to the signal processor 4.

[0061] Furthermore, in this embodiment, as Figure 1 As shown, since the imaging optical path overlaps with the excitation optical path and the fluorescence signal optical path, the imaging unit is also equipped with a second beam splitter 23. The illumination light emitted by the illumination module 21 is collected by the objective lens 22, reflected by the second beam splitter 23, and then focused by the imaging lens 24 onto the high-speed imaging camera 25. The excitation light emitted by the light source 31 passes through the beam shaping element 32, is reflected by the first beam splitter 33, transmits through the second beam splitter 23, and is then focused by the objective lens 22 onto the fluorescence signal monitoring site 16. The excited fluorescence is collected by the objective lens 22, transmits sequentially through the second beam splitter 23 and the first beam splitter 33, and is then focused by the converging lens 34 onto the high-speed detector 35.

[0062] Based on the imaging unit described in the above embodiments, when the signal processor 4 determines that the fluid currently passing through the fluorescence signal monitoring site 16 is a droplet based on the received fluorescence signal, the signal processor 4 sends a picture trigger command S3 to the high-speed imaging camera 25. The high-speed imaging camera 25 then acquires an image of the droplet at the imaging site 17 adjacent to the fluorescence signal monitoring site 16 according to the picture trigger command S3, and sends the image signal S2 to the signal processor 4. The signal processor 4 then determines whether the droplet is a droplet encapsulating a target cell based on the image signal S2.

[0063] In this embodiment, as Figure 1 and Figure 2 As shown, the sorting unit includes a voltage amplifier 41 and an electrode 42. The electrode 42 is disposed in the microfluidic chip 1. Specifically, the electrode 42 is located on the side of the main delivery channel 11 and near the sorting site 18. The voltage amplifier 41 is connected to the electrode 42 and the signal processor 4 respectively. Under the control of the signal processor 4, the voltage amplifier 41 applies a voltage to the electrode 42 to generate a non-uniform electric field, thereby sorting out the droplets encapsulating the target cells.

[0064] Specifically, when the signal processor 4 determines that the droplet is a droplet encapsulating the target cell based on the image signal, the signal processor 4 sends a sorting trigger command S4 to the voltage amplifier 41. The voltage amplifier 41 applies voltage to the electrode 42 according to the sorting trigger command S4, thereby forming a non-uniform electric field in the area where the sorting site 18 is located, and then generating a dielectrophoretic force to deflect the flow path of the droplet encapsulating the target cell, controlling the droplet encapsulating the target cell to flow out from the first output channel 14. When the signal processor 4 determines that the droplet does not encapsulate the target cell based on the judgment result of the image signal S2, the voltage amplifier 41 does not apply voltage to the electrode 42. At this time, the droplet and other fluids in the main delivery channel 11 flow out from the second output channel 15 according to the initial flow path, thereby realizing the sorting of the droplet encapsulating the target cell from other droplets and obtaining the droplet encapsulating the target cell.

[0065] The signal processor 4 is used to realize signal synchronization triggering between multiple systems, as well as real-time functions such as signal and image processing. The signal processor 4 is a microprocessor for real-time digital signal processing operations, having a signal input module and a signal output module, and can be implemented using dedicated standard circuits, dedicated integrated circuits, digital signal processors, and field-programmable gate array (FPGA) chips. In this invention, a FPGA chip is preferred. The signal input module of the signal processor 4 is connected to the high-speed imaging camera 25 in the imaging unit and the high-speed detector 35 in the fluorescence detection unit, respectively, for receiving image signal S2 and fluorescence signal S1. The signal output module of the signal processor 4 is connected to the high-speed imaging camera 25 in the imaging unit and the voltage amplifier 41 in the sorting unit, respectively, for issuing image trigger command S3 and sorting trigger command S4, realizing the functions of image triggering and sorting triggering. The signal processor 4 has the functions of detecting droplets from fluorescence signals and image processing, including image processing algorithms. The signal processor 4 can be connected to a computer to visualize the real-time processing process.

[0066] Based on the label-free live cell screening system provided in the above embodiments, this invention also provides a label-free live cell screening method based on droplet microfluidics, the method comprising the following steps:

[0067] I. System Debugging: Fix the microfluidic chip on the stage and adjust the stage's three-dimensional position so that the fluorescence signal monitoring site and imaging site of the microfluidic chip are within the objective lens's field of view. Simultaneously, adjust the excitation light intensity to locate the light spot and align it with the fluorescence signal monitoring site. The excitation light spot continuously excites the fluorescence of the fluid flowing through the fluorescence signal monitoring site. Adjust the illumination mode and the high-speed imaging camera's exposure time, frame rate, and other parameters, and put the high-speed imaging camera into standby mode, awaiting the signal processor's image capture trigger command. Adjust the voltage amplifier to standby mode, awaiting the signal processor's analysis trigger command.

[0068] It should be noted that the sorting site may not be within the field of view of the objective lens. However, since the distance between the fluorescence signal monitoring site, the imaging site, and the sorting site on the microfluidic channel is very small, the sorting site is usually within the field of view of the objective lens.

[0069] 2. The driving droplet, along with the continuous liquid phase, flows from the first end to the second end in the microfluidic channel of the microfluidic chip.

[0070] Specifically, the fluid drive device is connected to the channel containing the continuous phase and droplets (including droplets encapsulating target cells, droplets encapsulating non-target cells, and blank droplets without any cells). The pressure of the fluid drive device is adjusted so that the droplets continuously flow from the fluorescence signal monitoring site through the imaging site and the sorting site at fixed intervals, with the droplet passing frequency being no less than 1000 Hz.

[0071] 3. Control the fluorescence detection unit to emit excitation light to the fluorescence signal monitoring site to excite the fluorescence signal of the droplet, collect the fluorescence signal and feed it back to the signal processor.

[0072] Fourth, the signal processor determines whether a droplet passes through the fluorescence signal monitoring site based on the fluorescence signal: if so, it triggers the imaging unit to acquire an image of the droplet at the imaging site and feeds it back to the signal processor.

[0073] In a specific scheme, determining whether a droplet has passed through the fluorescence signal monitoring site includes: the signal processor continuously receives a fluorescence signal sequence fed back from the fluorescence detection unit, and compares the fluorescence signal sequence with a set fluorescence signal threshold: when the signal value of the fluorescence signal sequence first jumps from below the threshold to above the threshold and generates a rising edge, and then jumps from above the threshold to below the threshold and generates a falling edge, it is determined that a droplet has passed through the fluorescence signal monitoring site.

[0074] In other embodiments, a waveform shaping circuit (such as a Schmitt trigger) is connected to the output of the high-speed detector, and its output is connected to a signal processor or a high-speed imaging camera. When the droplet passes through, the waveform shaping circuit can directly output a rectangular square wave trigger signal, which can be used as a camera's image triggering method.

[0075] 5. The signal processor determines whether the droplet contains a target cell based on the image of the droplet: if so, it triggers the sorting unit to sort the droplet containing the target cell at the sorting point.

[0076] In a specific scheme, determining whether the cell droplet encapsulates the target cell based on the image includes: performing background filtering on the original image of the acquired droplet; applying morphological operators to process the filtered image to identify the droplet boundary and cells within the droplet, obtaining cell mask morphology data; calculating and acquiring the phenotypic features (e.g., density, morphology) of the mask-marked cells within the droplet and comparing them with preset conditions; if the phenotypic features meet the preset conditions, it is determined that the droplet encapsulates the target cell. It should be noted that the image processing function for the droplet in the signal processor is not limited to the algorithm described above; other algorithms capable of identifying cells within droplets can also be used.

[0077] In this embodiment of the invention, the signal processor is selected as a Field Programmable Gate Array (FPGA) chip. Using an FPGA-based hardware real-time image analysis method accelerates image analysis, increases droplet sorting frequency, and increases cell screening throughput. The image recognition algorithm, based on multi-dimensional information about cells encapsulated within droplets, can screen and classify cell populations with different phenotypes.

[0078] In a specific scheme, triggering the sorting unit to sort the droplets encapsulating target cells at the sorting point includes: the signal processor controlling the sorting unit to generate a non-uniform electric field at the sorting point, causing the flow path of the droplets encapsulating target cells to deviate, and controlling the droplets encapsulating target cells to flow through a predetermined output channel, thereby screening out the droplets encapsulating target cells.

[0079] In summary, the label-free live cell screening system and method based on droplet microfluidics provided in the above embodiments of the present invention uses excitation light to excite the fluorescence signal inherent in the cell droplets, and identifies and determines the droplets based on the fluorescence signal, which has the following beneficial effects:

[0080] (1) No need to use pinholes to limit the monitoring area, making hardware setup simpler; the excitation light will form a light spot in the imaging field of the high-speed imaging camera. As long as the microfluidic chip channel is aligned with the light spot, the monitoring of the microdroplets flowing through the channel can be realized. The device setup is more convenient and does not require frequent calibration.

[0081] (2) The monitoring of droplets and the imaging of high-speed imaging cameras use light signals of different wavelengths that do not interfere with each other. The intensity of the illumination source used for imaging can be adjusted at will as needed. Furthermore, the imaging modes of cells inside droplets are more diverse. In addition to bright field imaging, dark field, phase difference, differential interference phase difference (DIC) and other imaging modes can also be used, greatly expanding the application range.

[0082] (3) The signal-to-noise ratio of microdroplet monitoring signals is higher, and the signal is not affected by the contents of microdroplets, nor by the physical and chemical properties of microdroplets themselves. Therefore, the monitoring signal is more stable, thereby improving the sensitivity and accuracy of droplet monitoring, improving the reliability of cell screening, and also increasing the cell screening throughput. The cell droplet sorting frequency can reach more than 1000 Hz.

[0083] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A label-free live cell screening system based on droplet microfluidics, characterized in that, include: A microfluidic chip includes a microfluidic channel configured for transporting droplets; a fluorescence signal monitoring site, an imaging site, and a sorting site are sequentially arranged on the microfluidic channel. A fluorescence detection unit is configured to emit excitation light toward the fluorescence signal monitoring site to excite the fluorescence signal carried by the droplet, and to collect the fluorescence signal; An imaging unit is configured to acquire an image of a droplet at the imaging site using illumination light emitted therefrom; the illumination light and the excitation light have different wavelengths. The sorting unit is configured to sort droplets containing target cells at the sorting site; A signal processor is connected to the fluorescence detection unit, the imaging unit, and the sorting unit, respectively. When the signal processor detects a droplet passing through the fluorescence signal monitoring site based on the fluorescence signal, it triggers the imaging unit to acquire an image of the droplet at the imaging site. When the signal processor determines that the droplet contains a target cell based on the image, it triggers the sorting unit to sort the droplet containing the target cell at the sorting site. The target cell is an unlabeled target cell.

2. The label-free live cell screening system according to claim 1, characterized in that, The microfluidic channel includes a main delivery channel, a first injection channel and a second injection channel connected to a first end of the main delivery channel, and a first output channel and a second output channel connected to a second end of the main delivery channel; the fluorescence signal monitoring site, imaging site and sorting site are arranged sequentially on the main delivery channel from the first end to the second end, and the sorting site is located near the intersection of the first output channel and the second output channel.

3. The label-free live cell screening system according to claim 2, characterized in that, The microfluidic chip is connected to a fluid driving device, which is used to inject droplets into the first injection channel and inject a continuous liquid phase into the second injection channel, and drive the droplets to flow from the first end of the main delivery channel toward the second end.

4. The label-free live cell screening system according to claim 3, characterized in that, The fluorescence detection unit includes an excitation light module and a detection module. The excitation light module includes a light source and a beam shaping element. The detection module includes a converging lens and a high-speed detector. The excitation light emitted by the light source passes through the beam shaping element and is incident on the fluorescence signal monitoring site to excite fluorescence. The excited fluorescence is converged by the converging lens and incident on the high-speed detector. The high-speed detector sends the collected fluorescence signal to the signal processor.

5. The label-free live cell screening system according to claim 3, characterized in that, The imaging unit includes an illumination module, an objective lens, an imaging lens, and a high-speed imaging camera. The illumination module and the objective lens are located on opposite upper and lower sides of the microfluidic channel. The illumination light emitted by the illumination module is collected by the objective lens and focused onto the high-speed imaging camera via the imaging lens. The high-speed imaging camera sends the acquired image signal to the signal processor.

6. The label-free live cell screening system according to claim 3, characterized in that, The sorting unit includes a voltage amplifier and an electrode. The electrode is disposed in the microfluidic chip and is located on the side of the main delivery channel and near the sorting site. Under the control of the signal processor, the voltage amplifier applies a voltage to the electrode to generate a non-uniform electric field, thereby sorting out droplets encapsulating target cells.

7. A label-free live cell screening method based on droplet microfluidics, characterized in that, Using the label-free live cell screening system as described in any one of claims 1-6, the method includes the steps of: The driving droplet flows from the first end to the second end in the microfluidic channel of the microfluidic chip along with the continuous liquid phase; The fluorescence detection unit is controlled to emit excitation light toward the fluorescence signal monitoring site to excite the fluorescence signal of the droplet, and the fluorescence signal is collected and fed back to the signal processor; The signal processor determines whether a droplet has passed through the fluorescence signal monitoring site based on the fluorescence signal; if so, it triggers the imaging unit to acquire an image of the droplet at the imaging site and feeds it back to the signal processor. The signal processor determines whether the droplet contains a target cell based on the image of the droplet; if so, it triggers the sorting unit to sort the droplet containing the target cell at the sorting point.

8. The label-free live cell screening method according to claim 7, characterized in that, The determination of whether a droplet passes through the fluorescence signal monitoring site includes: The signal processor continuously receives the fluorescence signal sequence fed back from the fluorescence detection unit and compares the fluorescence signal sequence with a set fluorescence signal threshold: when the signal value of the fluorescence signal sequence first jumps from below the threshold to above the threshold and generates a rising edge, and then jumps from above the threshold to below the threshold and generates a falling edge, it is determined that a droplet has passed through the fluorescence signal monitoring site.

9. The label-free live cell screening method according to claim 7, characterized in that, The step of determining whether the droplet contains a target cell based on the image of the droplet includes: Background filtering is applied to the original images of the collected droplets; Morphological operators are applied to process the filtered image to identify droplet boundaries and cells within the droplets, thereby obtaining mask morphological data of the cells. The phenotypic features of the cells marked by the mask inside the droplet are calculated and compared with preset conditions. If the phenotypic features meet the preset conditions, it is determined that the droplet encapsulates the target cell.

10. The label-free live cell screening method according to claim 7, characterized in that, The triggering of the sorting unit to sort the droplets encapsulating the target cells at the sorting site includes: The signal processor controls the sorting unit to generate a non-uniform electric field at the sorting point, causing the flow path of the droplets encapsulating the target cells to deviate, and controls the droplets encapsulating the target cells to flow through a predetermined output channel, thereby screening out the droplets encapsulating the target cells.

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