CN114907960A - Label-free living cell screening system and method based on droplet microfluidics - Google Patents
Label-free living cell screening system and method based on droplet microfluidics Download PDFInfo
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Abstract
The invention provides a label-free living cell screening system and method based on droplet microfluidics, wherein the system comprises: the microfluidic chip is provided with a fluorescence signal monitoring site, an imaging site and a sorting site; a fluorescence detection unit configured to excite a fluorescence signal at a fluorescence signal monitoring site and collect the fluorescence signal; an imaging unit configured to acquire an image of the droplet at an imaging site; a sorting unit configured to sort out droplets encapsulating the target cells at a sorting site; the signal processor is used for triggering the imaging unit to collect the image of the liquid drop at the imaging site when the liquid drop passing through the fluorescent signal monitoring site is identified based on the fluorescent signal; and when the signal processor judges that the target cells are wrapped in the liquid drops based on image analysis, the sorting unit is triggered to sort the liquid drops wrapping the target cells at the sorting sites. The invention monitors the liquid drops by using the fluorescent signals, improves the monitoring efficiency and accuracy, and greatly improves the precision and flux of liquid drop sorting.
Description
Technical Field
The invention relates to the technical field of cell sorting, in particular to a system and a method for label-free living cell screening based on droplet microfluidics.
Background
The marker-free living cell screening means that the target cells are separated from one population and used for subsequent culture, analysis and utilization on the premise of ensuring the cell activity only according to the physiological phenotypic characteristics of the cells without any pretreatment on the cells. The label-free living cell screening has very wide application prospect in the field of biology, and particularly has great application potential in the aspect of screening microbial cells with important industrial or medical values. Droplet microfluidics is considered to be one of the best technical approaches for label-free live cell screening, mainly because a large number of micro-droplets can provide independent growth spaces for each cell in a population, the cells grow in the respective spaces and exhibit physiological phenotypes, macromolecular proteins or small molecular compounds secreted by the cells are confined in the respective droplet spaces, interference between the cells is completely eliminated, and cells with specific phenotypic characteristics are more easily identified and screened.
At present, the label-free living cell screening method based on droplet microfluidics can be divided into five categories according to the cell phenotype detection principle: light absorption/scattering, raman spectroscopy, mass spectrometry, electrochemical methods, imaging methods. Among them, raman spectroscopy, mass spectrometry and electrochemical methods can only detect cell metabolites, and thus have a limited range of application; the light absorption/scattering method can only detect the change of the cell number, and the application range is very limited; the imaging method can detect various physiological phenotypic characteristics including cell morphology, cell quantity and metabolic activity, and has wider application prospect. Therefore, the development of imaging-based droplet microfluidic label-free live cell screening technology is of great significance.
Imaging-based droplet microfluidic label-free live cell screening methods generally include the following steps:
(1) micro-fluidic chips are used for generating micro-droplets with uniform sizes, and single or multiple cells are wrapped in the micro-droplets while the micro-droplets are generated;
(2) collecting and incubating the microdroplets to allow cells within the microdroplets to grow and exhibit a physiological phenotype;
(3) injecting the micro-droplets into a micro-fluidic droplet sorting chip, collecting an image of each micro-droplet, and identifying target cells by analyzing physiological phenotypic characteristics of cells in the micro-droplets;
(4) after the target cells are identified, the micro liquid drops coated with the target cells are separated, the target cells are obtained by recovering the separated micro liquid drops, and finally the purpose of cell screening is achieved.
The core point of the above method is the acquisition of microdroplet images and the analysis of cell physiological phenotypic characteristics in step (3). Wherein, the collection and analysis speed of the micro-droplet image data directly determines the flux of cell screening; the quality of the microdroplet image and the performance of the cell physiological phenotypic characteristic analysis directly influence the sensitivity and the precision of cell screening. At present, a series of micro-droplet image acquisition and cell physiological phenotype characteristic analysis methods are proposed. These methods can be divided into two categories depending on the image acquisition mode:
a continuous image acquisition method comprises the following steps: continuously acquiring images of a microfluidic chip channel at a fixed frequency by using a high-speed camera, and analyzing whether micro-droplets flowing through the acquired images exist in real time; when micro liquid drops exist in the acquired image, further analyzing the physiological phenotype characteristics of the cells in the liquid drops, and judging whether the cells in the micro liquid drops are target cells or not; when the target cell is determined, a droplet sorting function is performed to recover the target cell.
In the continuous image capturing method, the image captured by the high-speed camera is mostly invalid data, that is, image data in which the droplet is not captured. However, in order to find the images of the captured droplets, these invalid data still need to be analyzed one by one, and the image analysis is the rate-limiting step of the whole technical method, and the analysis of a large amount of invalid image data seriously affects the performance efficiency of droplet sorting, and simultaneously limits the screening throughput of cells. In addition, analysis of invalid data wastes a large amount of hardware resources, and the equipment cost is higher on the premise of achieving the same cell screening effect. The droplet sorting frequency of the method can reach 10-30 Hz.
Secondly, a triggering type image acquisition method comprises the following steps: monitoring micro liquid drops flowing through a microfluidic chip channel by using a photoelectric detector, wherein when the liquid drops pass through the chip channel, the photoelectric detector can detect pulse-type change of the brightness, and at the moment, triggering a high-speed camera to acquire images of the micro liquid drops; then, carrying out real-time analysis on the image, identifying the outline of the droplet, analyzing the physiological phenotype characteristics of the cells in the droplet, and judging whether the cells in the micro-droplet are target cells or not; when the target cell is determined, a droplet sorting function is performed to recover the target cell.
The hardware structure mode of the triggering image acquisition method is as follows: the micro-fluidic chip is placed on an objective table of the inverted microscope, the high-speed camera is arranged on an imaging light path of the inverted microscope, and the micro-fluidic chip is illuminated by adopting transmitted light and generates a bright-field microscopic image; the light signal collected by the objective lens is divided into two parts by using the spectroscope, wherein one part of the light signal is transmitted to the high-speed camera for imaging, and the other part of the light signal is transmitted to the photoelectric detector for monitoring the liquid drops. The irregular pulse signals caused by the liquid drops passing are converted into pulse level signals and transmitted to the high-speed camera for triggering the image acquisition of the liquid drops; the output end of the high-speed camera is connected to a computer host, the image data is transmitted to the computer host, and the analysis of the image data is executed on the computer host; when the target cells are detected and the liquid drop sorting is required to be executed, the computer host sends sorting instructions to the equipment capable of generating sorting electric pulse signals, and finally the signals are transmitted to the microfluidic liquid drop sorting chip and the liquid drop sorting is executed. The droplet sorting frequency of this method can reach 40-100 Hz.
The existing trigger type image acquisition method realizes the monitoring of micro-droplets flowing through a channel of a micro-fluidic chip by detecting the intensity change of transmitted light. The method collects the image after monitoring the liquid drop, avoids collecting a large amount of invalid image data, but also brings new problems, and mainly comprises the following steps:
the microdroplet volume is small, and the transmitted light intensity change caused by the microdroplet is very weak, so that the signal-to-noise ratio of a microdroplet monitoring signal based on the principle is very low, the monitoring sensitivity and accuracy of the microdroplet are low, and the screening flux of the microdroplet is low. If it is desired to accurately detect the droplets, it is necessary to use Pinhole to narrow the detection range to a level close to the diameter of the droplets. The size selection and setting of the position of the Pinhole relative to the field of view of the high-speed camera are cumbersome, and the post-correction is also cumbersome.
The same illuminating light source is used for imaging of the high-speed camera and monitoring of the micro-droplets by the photoelectric detector, and in order to meet the requirements for imaging and monitoring of the droplets, the illumination intensity, the exposure time of the camera and the sensitivity of the photoelectric detector need to be adjusted at the same time, so that the three are highly matched, and the setting workload of each device is greatly increased. Meanwhile, in order to ensure the stability of the microdroplet monitoring signal, the intensity of the illumination light source needs to be fixed in the detection process, so that the intensity of the imaging illumination light cannot be adjusted at will according to different imaging requirements, and the flexibility of the method is greatly reduced.
The photodetector monitoring of the microdroplet signal is illuminated by means of transmitted light, so that imaging of the microdroplet and its internal cells can only be performed by means of bright field imaging based on transmitted light illumination, which greatly limits the scope of application of such methods.
Microdroplet monitoring based on transmitted light intensity is susceptible to a number of factors, such as: the change of light intensity can be caused by the substance components such as cells in the micro-droplets, the change of the change rate of optical signals can be caused by the difference of the refractive indexes of the interfaces of the micro-droplets formed by using different oil-water systems, and the like. These problems can cause the sensitivity and accuracy of microdroplet monitoring to be reduced, which in turn affects the reliability of cell screening.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a label-free living cell screening system and method based on droplet microfluidics, so as to solve the problem of how to improve the sensitivity and accuracy of droplet monitoring and further improve screening flux.
In order to solve the above problems, it is an aspect of the present invention to provide a label-free living cell screening system based on droplet microfluidics, the system comprising:
a microfluidic chip comprising 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 configured to emit excitation light to the fluorescence signal monitoring site to excite a fluorescence signal and collect the fluorescence signal;
an imaging unit configured for acquiring an image of the droplet at the imaging site;
a sorting unit configured for sorting out droplets encapsulating target cells at the sorting site;
the signal processor is respectively connected with the fluorescence detection unit, the imaging unit and the sorting unit;
the signal processor triggers the imaging unit to acquire an image of the liquid drop at the imaging site when the signal processor identifies that the liquid drop passes through the fluorescent signal monitoring site based on the fluorescent signal; and when the signal processor judges that the target cells are wrapped in the liquid drops on the basis of the image, the sorting unit is triggered to sort the liquid drops wrapping the target cells out at the sorting sites.
In a specific scheme, the microfluidic channel comprises a main conveying channel, a first injection channel and a second injection channel which are connected with a first end of the main conveying channel, and a first output channel and a second output channel which are connected with a second end of the main conveying channel; the fluorescence signal monitoring site, the imaging site and the sorting site are sequentially arranged on the main conveying channel from a first end to a second end, and the sorting site is arranged close to the junction of the first output channel and the second output channel.
In a specific scheme, the microfluidic chip is connected with a fluid driving device, and the fluid driving device is used for injecting droplets into the first injection channel and injecting a continuous liquid phase into the second injection channel, and driving the droplets to flow from the first end to the second end of the main conveying channel.
In a specific scheme, the fluorescence detection unit comprises an excitation light module and a detection module, wherein the excitation light module comprises a light source and a light beam shaping element, and the detection module comprises a converging lens and a high-speed detector; the fluorescence signal monitoring site is used for monitoring fluorescence signals, wherein excitation light emitted by the light source enters the fluorescence signal monitoring site after passing through the beam shaping element so as to excite fluorescence, the excited fluorescence is converged and enters the high-speed detector through the converging lens, and the high-speed detector sends acquired fluorescence signals to the signal processor.
In a specific scheme, the imaging unit comprises an illuminating module, an objective lens, an imaging lens and a high-speed imaging camera, the illuminating module and the objective lens are positioned on the upper side and the lower side of the microfluidic channel, illuminating light emitted by the illuminating module is collected by the objective lens, the illuminating light is focused on the high-speed imaging camera through the imaging lens, and the high-speed imaging camera sends acquired image signals to the signal processor.
In a specific scheme, the sorting unit comprises a voltage amplifier and electrodes, the electrodes are arranged in the microfluidic chip, the electrodes are positioned on the side surface of the main conveying channel and close to the sorting sites, and the voltage amplifier applies voltage to the electrodes under the control of the signal processor to generate a non-uniform electric field so as to sort out the liquid drops wrapping the target cells.
Another aspect of the present invention is to provide a label-free living cell screening method based on droplet microfluidics, using the label-free living cell screening system as described above, the method comprising the steps of:
driving the liquid drop to flow from the first end to the second end along with the continuous liquid phase in the microfluidic channel of the microfluidic chip;
controlling the fluorescence detection unit to emit exciting light to the fluorescence signal monitoring site so as to excite a fluorescence signal of the liquid drop, collecting the fluorescence signal and feeding the fluorescence signal back to the signal processor;
the signal processor judges whether liquid drops pass through the fluorescent signal monitoring site according to the fluorescent signal: if yes, triggering the imaging unit to acquire the image of the liquid drop at the imaging site and feeding the image back to the signal processor;
the signal processor judges whether the target cell is wrapped in the liquid drop according to the image: if yes, triggering the sorting unit to sort out the liquid drops wrapping the target cells at the sorting site.
In a specific embodiment, the determining whether any liquid drop 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 value: when the signal value of the fluorescence signal sequence firstly jumps from being lower than the threshold value to being higher than the threshold value and generates a rising edge, and then jumps from being higher than the threshold value to being lower than the threshold value and generates a falling edge, the situation that the liquid drop passes through the fluorescence signal monitoring site is judged.
In a specific embodiment, the determining whether the droplet wraps the target cell according to the image includes: carrying out background filtering on the acquired original image of the liquid drop; processing the filtered image by using a morphological operator, and identifying the liquid drop boundary and cells in the liquid drop to obtain mask morphological data of the cells; and calculating and acquiring the phenotypic characteristics of the cells of the mask marks in the liquid drops, comparing the phenotypic characteristics with preset conditions, and judging that the liquid drops wrap the target cells if the phenotypic characteristics meet the preset conditions.
In a specific embodiment, the triggering the sorting unit to sort the droplet wrapping the target cell at the sorting site includes: the signal processor controls the sorting unit to generate a non-uniform electric field at the sorting site, shifts the flow path of the target cell-encapsulated droplets, and controls the target cell-encapsulated droplets to flow from a predetermined output channel, thereby screening the target cell-encapsulated droplets.
The system and the method for screening the unmarked living cells based on the droplet microfluidics, which are provided by the embodiment of the invention, use the exciting light to excite the fluorescence signal carried by the droplet, and identify and judge the droplet based on the fluorescence signal, have the following beneficial effects:
(1) a pinhole (pinhole) is not needed to limit a monitoring area, and hardware is simpler to build; the exciting light can form the facula in the formation of image field of vision of high-speed camera, as long as aim at the facula with micro-fluidic chip channel can realize the monitoring to the interior tiny droplet of flow through of passageway, and the setting of equipment is more convenient, and does not need frequent calibration.
(2) The monitoring of the liquid drop and the high-speed camera imaging adopt optical signals with different wavelengths, mutual interference is avoided, the intensity of an illuminating light source for imaging can be adjusted at will according to needs, the imaging mode of cells in the liquid drop is more diversified, besides bright field imaging, various imaging modes such as dark field, phase difference and DIC can be used, and the application range is greatly expanded.
(3) The signal-to-noise ratio of the micro-droplet monitoring signal is higher, the signal is not influenced by the content of the micro-droplet, and is not influenced by the physical and chemical properties of the micro-droplet, so that the stability of the monitoring signal is higher, the sensitivity and the accuracy of droplet monitoring are improved, the reliability of cell screening is improved, the cell screening flux is also improved, and the sorting frequency of the cell droplets can reach more than 1000 Hz.
Drawings
FIG. 1 is a block diagram of a system for screening label-free living cells according to an embodiment of the present invention;
fig. 2 is a schematic structural view of a microfluidic channel in an embodiment of the present invention.
Detailed Description
To the accomplishment of the foregoing and related ends, the invention, then, comprises the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative aspects and implementations of the invention. These are indicative, however, of but a few of the various ways in which the principles of the invention may be employed. Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the drawings.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the scheme according to the present invention are shown in the drawings, and other details not so relevant to the present invention are omitted.
Aiming at the problem of low screening amount in the existing liquid drop microfluidic label-free living cell screening method based on imaging and the problems caused by the fact that a photoelectric detector for monitoring liquid drops and an imaging device for collecting liquid drop images use the same illumination light source in a triggered image collection method, the embodiment of the invention provides a liquid drop microfluidic label-free living cell screening system and method.
Based on the above concept, the embodiment of the present invention first provides a label-free living cell screening system based on droplet microfluidics, which includes a microfluidic chip, a fluorescence detection unit, an imaging unit, a sorting unit and a signal processor.
Wherein the microfluidic chip comprises at least a microfluidic channel configured for transporting droplets; and the microfluidic channel is sequentially provided with a fluorescence signal monitoring site, an imaging site and a sorting site. The fluorescence detection unit is configured to emit excitation light to the fluorescence signal monitoring site to excite a fluorescence signal, and to collect the fluorescence signal. The imaging unit is configured for acquiring image signals of the droplets at the imaging site. The sorting unit is configured for sorting out droplets encapsulating target cells at the sorting site; the signal processor is respectively connected with the fluorescence detection unit, the imaging unit and the sorting unit.
And the signal processor identifies and judges whether liquid drops pass through the fluorescent signal monitoring sites or not based on the fluorescent signals, and if so, the imaging unit is triggered to acquire images of the liquid drops at the imaging sites. The signal processor also judges whether the liquid drops wrap the target cells or not based on the image, and if so, the sorting unit is triggered to sort the liquid drops wrapping the target cells at the sorting sites.
In this embodiment, referring to fig. 1 and 2, the microfluidic chip 1 is disposed on the stage 2, a microfluidic channel 10 is disposed in the microfluidic chip 1, and the microfluidic channel 10 includes a main transport channel 11, a first injection channel 12 and a second injection channel 13 connected to a first end of the main transport channel 11, and a first output channel 14 and a second output channel 15 connected to a second end of the main transport channel 11. Wherein the first injection channel 12 is used for injecting droplets, and the second injection channel 13 is used for injecting a continuous liquid phase. The first output channel 14 is used for flowing out the sorted target cell-encapsulated droplets, and the second output channel 15 is used for flowing out other droplets besides the target cell-encapsulated droplets and other fluids. It should be noted that the droplets injected from the first injection channel 12 include droplets that wrap target cells, droplets that wrap non-target cells, and blank droplets that do not wrap any cells.
Further, as shown in fig. 2, a fluorescence signal monitoring site 16, an imaging site 17, and a sorting site 18 are sequentially provided on the main transport path 11, the fluorescence signal monitoring site 16, the imaging site 17, and the sorting site 18 are sequentially provided on the main transport path 11 in a direction from the first end to the second end of the main transport path 11 (i.e., a flow direction of droplets), and the sorting site 18 is provided adjacent to a junction of the first output path 14 and the second output path 15.
Further, as shown in fig. 1, a fluid driving device 3 is connected to the microfluidic chip 1, and the fluid driving device 3 is configured to inject a droplet into the first injection channel 12 and a continuous liquid phase into the second injection channel 13, and drive the droplet to flow from the first end toward the second end of the main transport channel 11.
In this embodiment, the fluorescence detection unit includes an excitation light module and a detection module. As shown in fig. 1, the excitation light module comprises a light source 31 and a beam shaping element 32, and the detection module comprises a converging lens 34 and a high-speed detector 35. Wherein, the excitation light emitted from the light source 31 passes through the beam shaping element 32 and then enters the fluorescence signal monitoring site 16 to excite the fluorescence, and the excited fluorescence is converged and enters the high-speed detector 35 via the converging lens 34. The high-speed detector 35 is connected to the signal processor 4, and the high-speed detector 35 transmits the collected fluorescence signal S1 to the signal processor 4.
The light source 31 is provided by a laser or a light emitting diode light source, and is selected to be a laser in this embodiment, that is, the excitation light is laser light. The beam shaping element 32 is configured to shape the excitation light emitted by the light source 31 into a specific light beam, and the beam shaping element 32 may be selected from a collimator configured to collimate the light beam into parallel light or a cylindrical lens group configured to shape a circular light spot into a linear light spot, in this embodiment, the cylindrical lens group is selected, so that a signal-to-noise ratio of signal detection may be improved. In other embodiments, the beam shaping element 32 is not limited to the collimator and the set of cylindrical mirrors described above.
Further, in the present embodiment, as shown in fig. 1, since there is a portion where the optical path of the excitation light overlaps with the optical path of the fluorescence signal, a first beam splitter 33 is further disposed in the fluorescence detection unit, the excitation light emitted by the light source 31 passes through the beam shaping element 32, is reflected by the first beam splitter 33, and is incident on the fluorescence signal monitoring site 16 to excite fluorescence, and the excited fluorescence is transmitted through the first beam splitter 33 and is converged by the converging lens 34 and is incident on the high-speed detector 35.
Based on the fluorescence detection unit described in the above embodiment, the excitation light module therein continuously emits excitation light to the fluorescence signal monitoring site 16 to continuously excite fluorescence, and the detection module continuously collects fluorescence signals. When the liquid drop passes through the fluorescence signal monitoring site 16 along with the continuous liquid phase in the main conveying channel 11, the fluorescence signal excited from the liquid drop is different from the fluorescence signal excited from other fluids, so that the signal processor 4 can identify whether the liquid drop currently passes through the fluorescence signal monitoring site 16 or not based on the received fluorescence signal, realize the monitoring of the liquid drop, and further determine whether an imaging unit needs to be triggered to shoot to acquire image information according to the monitoring result.
In the present embodiment, as shown in fig. 1, 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 two opposite sides of the microfluidic chip 1, specifically, on two opposite sides of the microfluidic channel 10, as shown in fig. 1, the illumination module 21 is located on the upper side of the microfluidic chip 1, and the objective lens 22 is located on the lower side of the microfluidic chip 1.
Specifically, the illumination light emitted from the illumination module 21 is collected by the objective lens 22, and focused on the high-speed imaging camera 25 via the imaging lens 24. The high-speed imaging camera 25 is connected with the signal processor 4, the signal processor 4 controls the high-speed imaging camera 25 to capture images, and the high-speed imaging camera 25 sends captured image signals S2 to the signal processor 4.
Further, in the present embodiment, as shown in fig. 1, since there are overlapping portions of the imaging optical path, the excitation light optical path and the fluorescence signal optical path, a second beam splitter 23 is further disposed in the imaging unit, and the illumination light emitted from the illumination module 21 is collected by the objective lens 22, reflected by the second beam splitter 23, and focused on the high-speed imaging camera 25 by the imaging lens 24. The excitation light emitted from the light source 31 passes through the beam shaping element 32, is reflected by the first beam splitter 33, then is transmitted through the second beam splitter 23, is focused by the objective lens 22 and enters the fluorescence signal monitoring site 16, and the excited fluorescence is collected by the objective lens 22, sequentially passes through the second beam splitter 23 and the first beam splitter 33, and then is focused by the focusing lens 34 and enters the high-speed detector 35.
Based on the imaging unit described in the above embodiment, when the signal processor 4 determines that the fluid currently passing through the fluorescent signal monitoring site 16 is a droplet based on the received fluorescent signal, the signal processor 4 sends a photographing triggering instruction S3 to the high-speed imaging camera 25, the high-speed imaging camera 25 collects an image of the droplet at the imaging site 17 adjacent to the fluorescent signal monitoring site 16 according to the photographing triggering instruction S3 and sends the image signal S2 to the signal processor 4, and the signal processor 4 determines whether the droplet is a droplet encapsulating a target cell according to the image signal S2.
In this embodiment, as shown in fig. 1 and fig. 2, the sorting unit includes a voltage amplifier 41 and an electrode 42, the electrode 42 is disposed in the microfluidic chip 1, the electrode 42 is specifically located on a side of the main transport channel 11 and adjacent to the sorting site 18, the voltage amplifier 41 is respectively connected to the electrode 42 and the signal processor 4, and the voltage amplifier 41 applies a voltage to the electrode 42 under the control of the signal processor 4 to generate a non-uniform electric field, so as to sort out droplets that wrap target cells.
Specifically, when the signal processor 4 determines that the droplet is a droplet encapsulating the target cell according to the image signal, the signal processor 4 sends a sorting trigger instruction S4 to the voltage amplifier 41, and the voltage amplifier 41 applies a voltage to the electrode 42 according to the sorting trigger instruction S4 so as to form a non-uniform electric field in the region of the sorting site 18, thereby generating a dielectrophoretic force to shift the flow path of the droplet encapsulating the target cell and controlling the droplet encapsulating the target cell to flow out of the first output channel 14; on the other hand, when the signal processor 4 determines that the target cell is not encapsulated in the droplet based on the determination result of the image signal S2, the voltage amplifier 41 does not apply the voltage to the electrode 42, and the droplet and the other fluid in the main transport channel 11 flow out from the second output channel 15 according to the initial flow path, thereby achieving the separation of the droplet encapsulated with the target cell and obtaining the droplet encapsulated with the target cell by screening.
The signal processor 4 is configured to implement signal synchronization triggering among multiple systems, and real-time functions such as signal and image processing. The signal processor 4 is a microprocessor for real-time digital signal processing operation, has a signal input module and a signal output module, and can be realized by a special standard circuit, a special integrated circuit, a digital signal processor, a field programmable gate array chip and the like. A Field Programmable Gate Array (FPGA) chip is preferred in the present invention. 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 the image signal S2 and the fluorescence signal S1. The signal output module of the signal processor 4 is respectively connected to the high-speed imaging camera 25 in the imaging unit and the voltage amplifier 41 in the sorting unit, and is configured to send a photographing triggering instruction S3 and a sorting triggering instruction S4, so as to implement the functions of photographing triggering and sorting triggering. The signal processor 4 has functions of detecting liquid droplets from the fluorescent signal and image processing, including an image processing algorithm. The signal processor 4 may be arranged in connection with a computer for visually presenting the real-time processing.
Based on the label-free living cell screening system provided by the above embodiment, the embodiment of the invention also provides a label-free living cell screening method based on droplet microfluidics, and the method comprises the following steps:
firstly, debugging work of a system: fixing the micro-fluidic chip on an objective table, adjusting the three-dimensional position of the objective table to enable the fluorescent signal monitoring site and the imaging site of the micro-fluidic chip to be positioned in the visual field of the objective lens, and adjusting the intensity of exciting light to find a light spot site to enable the light spot site to be aligned with the fluorescent signal monitoring site. The excitation light spot continuously excites fluorescence of fluid flowing through the fluorescence signal monitoring site. And adjusting parameters such as an illumination mode and exposure time, frame rate and the like of the high-speed imaging camera, enabling the high-speed imaging camera to be in a standby state, and waiting for a photographing triggering instruction of the signal processor. And adjusting the voltage amplifier to be in a standby state, and waiting for an analysis trigger instruction of the signal processor.
It should be noted that the sorting sites may not be in the field of view of the objective lens, but because the spacing between the fluorescence signal monitoring sites, the imaging sites, and the sorting sites on the microfluidic channel is small, the sorting sites are usually in the field of view of the objective lens.
And secondly, driving the liquid drop to flow from the first end to the second end along with the continuous liquid phase in the microfluidic channel of the microfluidic chip.
Specifically, the fluid driving device is connected to channels for loading the continuous phase and droplets (including a droplet coated with a target cell, a droplet coated with a non-target cell, and a blank droplet not coated with any cell), respectively, and the pressure of the fluid driving device is adjusted so that the droplets continuously flow from the fluorescence signal monitoring site through the imaging site and the sorting site at intervals of a fixed distance, and the droplet passing frequency is not less than 1000 Hz.
And thirdly, controlling the fluorescence detection unit to emit exciting light to the fluorescence signal monitoring site so as to excite the fluorescence signal of the liquid drop, collecting the fluorescence signal and feeding the fluorescence signal back to the signal processor.
Fourthly, the signal processor judges whether liquid drops pass through the fluorescent signal monitoring site according to the fluorescent signal: and if so, triggering the imaging unit to acquire the image of the liquid drop at the imaging site and feeding the image back to the signal processor.
In a specific embodiment, the determining whether any liquid drop 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 value: when the signal value of the fluorescence signal sequence firstly jumps from being lower than the threshold value to being higher than the threshold value and generates a rising edge, and then jumps from being higher than the threshold value to being lower than the threshold value and generates a falling edge, the situation that the liquid drop passes through the fluorescence signal monitoring site is judged.
In other embodiments, a waveform shaping circuit (such as a schmitt trigger) is connected to the output of the high-speed detector, and the output of the waveform shaping circuit is connected to the signal processor or the high-speed imaging camera. When the liquid drop passes through, the waveform shaping circuit can directly output a trigger signal of a rectangular square wave to be used as a photographing trigger mode of the camera.
And fifthly, the signal processor judges whether the target cells are wrapped in the liquid drops according to the images of the liquid drops: and if so, triggering the sorting unit to sort out the droplets wrapping the target cells at the sorting site.
In a specific embodiment, the determining whether the cell droplet wraps the target cell according to the image includes: carrying out background filtering on the acquired original image of the liquid drop; processing the filtered image by using a morphological operator, and identifying the liquid drop boundary and cells in the liquid drop to obtain mask morphological data of the cells; and calculating and acquiring the phenotypic characteristics (such as density, morphology and the like) of the cells of the mask marks in the liquid drops, comparing the phenotypic characteristics with preset conditions, and judging that the liquid drops wrap the target cells if the phenotypic characteristics meet the preset conditions. It should be noted that the image processing function for the droplets in the signal processor is not limited to the above-mentioned algorithm, and other algorithms capable of performing cell recognition in corresponding droplets may be used.
The signal processor in the embodiment of the invention is selected as a Field Programmable Gate Array (FPGA) chip, and the FPGA-based hardware real-time image analysis method is used, so that the image analysis speed is increased, the liquid drop sorting frequency can be improved, and the cell screening flux is increased. The image recognition algorithm can screen and classify cell populations with different phenotypes based on multi-dimensional information of cells wrapped in the liquid drops.
In a specific embodiment, the triggering the sorting unit to sort the droplet wrapping the target cell at the sorting site includes: the signal processor controls the sorting unit to generate a non-uniform electric field at the sorting site, shifts the flow path of the target cell-encapsulated droplets, and controls the target cell-encapsulated droplets to flow from a predetermined output channel, thereby screening the target cell-encapsulated droplets.
In summary, the system and the method for screening label-free living cells based on droplet microfluidics provided by the above embodiments of the present invention use the excitation light to excite the fluorescence signal carried by the cell droplet, and identify and determine the droplet based on the fluorescence signal, which has the following beneficial effects:
(1) a pinhole (pinhole) is not needed to limit a monitoring area, and hardware is simpler to build; the exciting light can form a light spot in the imaging visual field of the high-speed imaging camera, the micro-fluidic chip channel can be monitored through flowing through the micro-droplet in the channel only by aligning the micro-fluidic chip channel to the light spot, the equipment is more convenient to set, and frequent calibration is not needed.
(2) The monitoring of the liquid drop and the imaging of the high-speed imaging camera adopt optical signals with different wavelengths, mutual Interference is avoided, the intensity of an illuminating light source for imaging can be randomly adjusted according to needs, the imaging mode of cells in the liquid drop is more diversified, besides bright field imaging, various imaging modes such as dark field, phase difference and Differential Interference Contrast (DIC) can be used, and the application range is greatly expanded.
(3) The signal-to-noise ratio of the micro-droplet monitoring signal is higher, the signal is not influenced by the content of the micro-droplet, and is not influenced by the physical and chemical properties of the micro-droplet, so that the stability of the monitoring signal is higher, the sensitivity and the accuracy of droplet monitoring are improved, the reliability of cell screening is improved, the cell screening flux is also improved, and the sorting frequency of the cell droplets can reach more than 1000 Hz.
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (10)
1. A label-free live cell screening system based on droplet microfluidics, comprising:
a microfluidic chip comprising 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 configured to emit excitation light to the fluorescence signal monitoring site to excite a fluorescence signal and collect the fluorescence signal;
an imaging unit configured for acquiring an image of the droplet at the imaging site;
a sorting unit configured for sorting out droplets encapsulating target cells at the sorting site;
the signal processor is respectively connected with the fluorescence detection unit, the imaging unit and the sorting unit;
the signal processor triggers the imaging unit to acquire an image of the liquid drop at the imaging site when the signal processor identifies that the liquid drop passes through the fluorescent signal monitoring site based on the fluorescent signal; and when the signal processor judges that the target cells are wrapped in the liquid drops on the basis of the image, the sorting unit is triggered to sort the liquid drops wrapping the target cells out at the sorting sites.
2. The label-free living cell screening system of claim 1, wherein the microfluidic channel comprises a main transport channel, a first injection channel and a second injection channel connected to a first end of the main transport channel, a first output channel and a second output channel connected to a second end of the main transport channel; the fluorescence signal monitoring site, the imaging site and the sorting site are sequentially arranged on the main conveying channel from a first end to a second end, and the sorting site is arranged close to the junction of the first output channel and the second output channel.
3. The label-free living cell screening system according to claim 2, wherein a fluid driving device is connected to the microfluidic chip, and the fluid driving device is configured to inject the droplet into the first injection channel and the continuous liquid phase into the second injection channel, and drive the droplet to flow from the first end toward the second end of the main transport channel.
4. The label-free living cell screening system according to claim 3, wherein the fluorescence detection unit comprises an excitation light module and a detection module, the excitation light module comprises a light source and a beam shaping element, and the detection module comprises a converging lens and a high-speed detector; the fluorescence signal monitoring site is used for monitoring fluorescence signals, wherein excitation light emitted by the light source enters the fluorescence signal monitoring site after passing through the beam shaping element so as to excite fluorescence, the excited fluorescence is converged and enters the high-speed detector through the converging lens, and the high-speed detector sends acquired fluorescence signals to the signal processor.
5. The system for screening unmarked living cells according to claim 3, wherein the imaging unit comprises an illumination module, an objective lens, an imaging lens and a high-speed imaging camera, the illumination module and the objective lens are positioned at the 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 on the high-speed imaging camera through the imaging lens, and the high-speed imaging camera sends the collected image signal to the signal processor.
6. The label-free living cell screening system of claim 3, wherein the sorting unit comprises a voltage amplifier and electrodes, the electrodes are arranged in the microfluidic chip, the electrodes are positioned on the side of the main conveying channel and adjacent to the sorting site, and the voltage amplifier applies a voltage to the electrodes under the control of the signal processor to generate a non-uniform electric field, so that droplets wrapping target cells are sorted out.
7. A label-free living cell screening method based on droplet microfluidics, using the label-free living cell screening system according to any one of claims 1 to 6, the method comprising the steps of:
driving the liquid drop to flow from the first end to the second end along with the continuous liquid phase in the microfluidic channel of the microfluidic chip;
controlling the fluorescence detection unit to emit exciting light to the fluorescence signal monitoring site so as to excite a fluorescence signal of the liquid drop, collecting the fluorescence signal and feeding the fluorescence signal back to the signal processor;
the signal processor judges whether liquid drops pass through the fluorescent signal monitoring site according to the fluorescent signal: if yes, triggering the imaging unit to acquire the image of the liquid drop at the imaging site and feeding the image back to the signal processor;
the signal processor judges whether target cells are wrapped in the liquid drops according to the images of the liquid drops: and if so, triggering the sorting unit to sort out the droplets wrapping the target cells at the sorting site.
8. The method of claim 7, wherein the determining whether any liquid drop passes through the fluorescence signal monitoring site comprises:
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 value: when the signal value of the fluorescence signal sequence firstly jumps from being lower than the threshold value to being higher than the threshold value and generates a rising edge, and then jumps from being higher than the threshold value to being lower than the threshold value and generates a falling edge, the situation that the liquid drop passes through the fluorescence signal monitoring site is judged.
9. The method for screening label-free living cells according to claim 7, wherein the determining whether the droplet is encapsulated with the target cell based on the image of the droplet comprises:
carrying out background filtering on the acquired original image of the liquid drop;
processing the filtered image by using a morphological operator, and identifying the liquid drop boundary and cells in the liquid drop to obtain mask morphological data of the cells;
and calculating and acquiring the phenotypic characteristics of the cells of the mask marks in the liquid drops, comparing the phenotypic characteristics with preset conditions, and judging that the liquid drops wrap the target cells if the phenotypic characteristics meet the preset conditions.
10. The method of claim 7, wherein the triggering the sorting unit to sort the droplets encapsulating the target cells at the sorting site comprises:
the signal processor controls the sorting unit to generate a non-uniform electric field at the sorting site, shifts the flow path of the target cell-encapsulated droplets, and controls the target cell-encapsulated droplets to flow from a predetermined output channel, thereby screening the target cell-encapsulated droplets.
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