FLUIDIC FLOW CYTOMETRY DEVICES AND PARTICLE SENSING BASED ON SIGNAL-ENCODING
Microfluidic devices, systems and techniques in connection with particle sorting in liquid, including cytometry devices and techniques and applications in chemical or biological testing and diagnostic measurements.
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This patent document is a continuation of U.S. patent application Ser. No. 15/722,550, with a filing date of Oct. 2, 2017, which is continuation of U.S. patent application Ser. No. 14/853,765, with a filing date of Sep. 14, 2015, now U.S. Pat. No. 9,778,164, which is a continuation of U.S. patent application Ser. No. 13/254,851, with a filing date of Nov. 28, 2011, now U.S. Pat. No. 9,134,221, issued on Sep. 15, 2015, which is a U.S. National Stage application under 35 U.S.C. § 371 of International Application No. PCT/US2010/026884, filed on Mar. 10, 2010, which claims priorities of U.S. Provisional Application No. 61/158,969 entitled “Particle sorter and sorting system with piezoelectric actuation” and filed Mar. 10, 2009, and U.S. Provisional Application No. 61/262,787 entitled “Flow cytometry system and method employing color-space-time coding” and filed Nov. 19, 2009, the entire disclosures of which are incorporated by reference as part of the disclosure of this document.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under RR024453, RR031424, and RR032225 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUNDThis patent document relates to fluidic devices and techniques in connection with particle sorting in liquid, including cytometry devices and techniques and applications in chemical or biological testing and diagnostic measurements.
Flow cytometry (FC) devices and systems can be used to characterize and analyze particles in liquid, e.g., physical and biochemical properties of cells and biochemical molecules or molecule clusters based on their optical responses as they are interrogated by external light sources in a serial manner. optical signals from such particles can be collected by an optical detector, such as a photomultiplier tube (PMT), and are analyzed or processed to extract information carried by the optical signals on the particles. The optical signals from the particles can be caused by one or more interactions between the input light and the particles such as forward scattering (FSC), side scattering (SSC), and fluorescence.
Cell sorting, including cell sorting at the single-cell level, has become an essential feature in the field of flow cytometry as researchers and clinicians become more interested in studying and purifying certain cells such as stem cells, circulating tumor cells, and rare bacteria species. Cell sorting can be achieved by various techniques. One example is applying vibrations to the jet flow from the nozzle to cause breakage of jet flow into droplets and subsequently using electrically charged plates to deflect cell-containing droplets into their respective collection tubes (droplets of no interest flow straight down to the waste tube without deflection).
Flow cytometry (FC) devices and systems can be implemented based on microfluidic technologies for research assays and diagnostics as well as for clinical applications. Microfluidic technologies range from simple microfluidic channels to complex microfludic devices that can mix fluids, pump liquids, perform digital logic, individually culture cells, determine optimal reaction conditions, and much more. Small-scale fluidic devices have low Reynolds numbers and can be used to achieve controlled laminar flow systems. Microfluidics further offers the advantages of small size for miniaturization and parallelization of devices. The compact size of microfludic devices opens the door to the potential of portable devices. Additionally, various fabrication processes for microfludic devices are suitable for mass production which can reduces the cost of such devices. Advances in microfludic devices can lead to low-cost lab-on-a-chip devices, useful tools to researchers, clinical laboratories, and point-of-care clinicians in remote and/or resource-poor settings.
SUMMARYThis document describes, among others, examples and implementations of techniques and devices for particle sorting in liquid, including cytometry devices and techniques.
In one aspect, a method for providing optical interrogation for flow cytometry includes directing light to one or more of the fluidic channels that are coupled to form a network of fluidic channels to illuminate fluid carried by the fluidic channels; providing optical signal structures that are respectively coupled to at least some of the fluidic channels at different locations to produce optical signals from the light illuminating the fluid, each optical signal carrying information indicative of a property of a particle carried in the fluid at a location of the respective optical signal structure, the optical signal structures being structured to produce, respectively, unique codes in the optical signals that are different from one another; using a single optical detector to collect light from all the optical signals generated at the optical signal structures to produce an electrical detector signal in response to the collected light; and processing the electrical detector signal based on the unique codes in the optical signals to separate information carried by the optical signals to extract information carried by each of the optical signals.
In another aspect, a system for flow cytometry is provided to include an input fluidic channel including a first port for receiving a sample fluid and a second port for outputting the received sample fluid; a particle sorting junction coupled to the second port of the input fluidic channel; branch fluidic channels coupled to the particle sorting junction as outlets of the sample liquid from the second port of the input fluidic channel; an actuator coupled to the particle sorting junction to control a direction of the sample fluid in the particle sorting junction in response to a sorting control signal, the actuator structured to interact with the sample fluid to change the direction of sample fluid to be in different directions corresponding to the branch fluidic channels, respectively, in response to changes in the sorting control signal, wherein the actuator is operable to direct a target particle in the sample fluid into a selected one of the branch fluidic channels; a particle detection module coupled to the input fluidic channel to receive light from the sample fluid in the input fluid channel, the particle detection module including an encoding structure that produces different optical signals from the received light and encodes the different optical signals with different codes; an optical detector that receives the different optical signals to produce a detector signal that carries information of the different optical signals and the different codes; and a particle sorter control module in communication with the particle detection module to receive the detector signal and in communication with the actuator to send the sorting control signal to the actuator, the particle sorter control module including a signal processing mechanism to extract information of the different optical signals from the detector signal based on the different codes in the different optical signals, and a control mechanism that produces the sorting control signal based on the extracted information, including timing of a particle detected at the particle detection module for arriving at the particle sorting junction.
In another aspect, a system for flow cytometry is provided to include an input fluidic channel structured to carry a sample fluid; a particle sorting junction coupled to one end of the input fluidic channel to receive a sample fluid from the input fluidic channel; branch fluidic channels coupled to the particle sorting junction as outlets of the sample liquid from the second port of the input fluidic channel; an actuator coupled to the particle sorting junction to control a direction of the sample fluid in the particle sorting junction in response to a sorting control signal, the actuator structured to interact with the sample fluid to change the direction of sample fluid to be in different directions corresponding to the branch fluidic channels, respectively, in response to changes in the sorting control signal, wherein the actuator is operable to direct a target particle in the sample fluid into a selected one of the branch fluidic channels; a particle detection module coupled to the input fluidic channel to receive light from the sample fluid in the input fluidic channel, the particle detection module structured to produce one or more first optical signals from the received light indicative of at least a speed of a particle in the sample fluid detected by the particle detection module; a branch verification structure coupled to one of the branch fluidic channels to receive light from the one branch fluidic channel and to produce a branch verification optical signal that can be used to verify whether a target particle is directed by the actuator into the one branch fluidic channel; an optical detector located to receive light which includes at least the one or more first optical signals from the particle detection module and the branch verification optical signal and to produce a detector signal that carries information contained in the received light; and a particle sorter control module in communication with the particle detection module to receive the detector signal and in communication with the actuator to send the sorting control signal to the actuator, the particle sorter control module including a signal processing mechanism to extract information from the detector signal, including timing of a particle detected at the particle detection module for arriving at the particle sorting junction based on information on the speed of the particle carried by the detector signal, and a control mechanism that produces the sorting control signal based on the extracted information, including the timing of the particle for arriving at the particle sorting junction. The signal processing mechanism in the particle sorter control module extracts information of the branch verification optical signal to produce an indicator that verifies whether a target particle is directed by the actuator into the one branch fluidic channel.
In another aspect, a system for flow cytometry is provided to include an input fluidic channel structured to carry a sample fluid; a particle sorting junction coupled to one end of the input fluidic channel to receive a sample fluid from the input fluidic channel; branch fluidic channels coupled to the particle sorting junction as outlets of the sample liquid from the second port of the input fluidic channel; an actuator coupled to the particle sorting junction to control a direction of the sample fluid in the particle sorting junction in response to a sorting control signal, the actuator structured to interact with the sample fluid to change the direction of sample fluid to be in different directions corresponding to the branch fluidic channels, respectively, in response to changes in the sorting control signal, wherein the actuator is operable to direct a target particle in the sample fluid into a selected one of the branch fluidic channels; a particle detection module coupled to the input fluidic channel to receive light from the sample fluid in the input fluidic channel, the particle detection module structured to produce one or more first optical signals from the received light indicative of at least a speed of a particle in the sample fluid detected by the particle detection module, each first optical signal being encoded with a unique first code; a branch optical signal structure coupled to one of the branch fluidic channels to receive light from the one branch fluidic channel and to produce a second optical signal that indicates a property of a particle in the one branch fluidic channel and is encoded with a unique second code that is different from each first code; an optical detector located to receive light which includes at least the one or more first optical signals from the particle detection module and the second optical signal and to produce a detector signal that carries information contained in the received light; and a particle sorter control module in communication with the particle detection module to receive the detector signal and in communication with the actuator to send the sorting control signal to the actuator, the particle sorter control module including a signal processing mechanism to extract information from the detector signal, including timing of a particle detected at the particle detection module for arriving at the particle sorting junction based on information on the speed of the particle carried by the detector signal, and a control mechanism that produces the sorting control signal based on the extracted information, including the timing of the particle for arriving at the particle sorting junction. The signal processing mechanism of the particle sorter control module separates information carried by the one or more first optical signals and the second optical signal based on the first codes and the second code.
In another aspect, a system for flow cytometry is provided to include fluidic channels structured to carry fluid and to form a network of fluidic channels; a light coupling structure to couple light into one or more of the fluidic channels to illuminate the fluid therein; optical signal structures that are respectively coupled to at least some of the fluidic channels at different locations to produce optical signals from the light illuminating the fluid, each optical signal carrying information indicative of a property of a particle carried in the fluid at a location of the respective optical signal structure, optical signal structures being structured to produce, respectively, unique codes in the optical signals that are different from one another; an optical detector that is positioned to receive the optical signals from the optical signal structures and to produce a detector signal based on received light of the optical signals; and a processing circuit coupled to the optical detector to receive the detector signal, the processing circuit separating information carried by the optical signals based on the unique codes to extract information carried by each of the optical signals.
In another aspect, a particle sorter for sorting particles in a fluid is provided to include a structure having an input channel connected at an actuation area to a plurality of output channels, wherein the particles in the fluid flow through the input channel to the actuation area, and each particle travels from the actuation area to one of the plurality of output channels, and a piezoelectric actuator for causing a flow disturbance in the actuation area in response to a control signal, wherein the flow disturbance operates to direct a particle along a trajectory to one of the plurality of output channels which is different than the output channel to which the particle would travel without the flow disturbance.
In another aspect, a particle sorting system for sorting particles of interest from other particles in a fluid is provided to include a structure having at least one input channel connected at an actuation area to a plurality of output channels, wherein the fluid flows through the input channel to the actuation area, and each particle travels from the actuation area to one of the plurality of output channels, a spatial filter having one or more slots and coupled to the input channel, a detector for generating a detection signal over time indicative of light emitted or scattered from a particle of interest, which light has passed through the one or more slots of the spatial filter, a processor in communication with the detector for analyzing the detection signal over time and generating a presence signal indicative of the presence of a particle of interest, a driver in communication with the processor and generating a control signal in response to the presence signal, and a piezoelectric actuator for causing a flow disturbance in the actuation area in response to the control signal, wherein the flow disturbance operates to direct a detected particle of interest along a trajectory to one of the plurality of output channels which is different than the output channel to which the particle would travel without the flow disturbance.
In another aspect, a particle sorting system for sorting particles of interest from other particles in a fluid includes a structure having at least one input channel connected at an actuation area to a plurality of output channels, wherein the fluid flows through the input channel to the actuation area, and each particle travels from the actuation area to one of the plurality of output channels, a spatial filter having one or more slots and coupled to the input channel, a detection unit that detects a particle of interest at a predetermined location and generating a control signal in response, and a piezoelectric actuator for causing a flow disturbance in the actuation area in response to the control signal. The flow disturbance operates to direct a detected particle of interest along a trajectory to one of the plurality of output channels which is different than the output channel to which the particle would travel without the flow disturbance.
In another aspect, a system for flow cytometry is provided to include a microfluidic channel; a first light conveying structure configured to convey substantially all visible light components, and having a first end proximate the microfluidic channel; at least one second light conveying structure having at least one second end proximate the microfluidic channel, and extending substantially alongside the first light conveying structure, wherein the at least one second light conveying structure is configured to convey at least one subset of the visible light components; and a light sensing device arranged proximate respective additional ends of each of the light conveying structures, the respective additional ends being respectively opposite the respective first and second ends. Respective portions of light emanating from material passing through the microfluidic channel are received by the respective light conveying structures and communicated at least in part to thereby to the light sensing device. An indication of the material passing through the microfluidic channel can be determined based upon one or more signals output by the light sensing device.
In another aspect, a method of performing flow cytometry is provided to include injecting first light into a microfluidic channel through which material is passing; receiving second light from the microfluidic channel into a plurality of waveguides, wherein a first of the waveguides is conductive of substantially all visible light components, and a second of the waveguides is conductive of a subset of the visible light components; and conveying first and second portions of the second light through the first and second waveguides from respective first ends of the waveguides to respective second ends of the waveguides; and communicating at least some of each of the conveyed first and second portions of the second light to a photodetector; and outputting a color-space-time signal from the photodetector.
In yet another aspect, a method of performing flow cytometry is provided to include injecting first light into a microfluidic channel through which material is passing; receiving second light from the microfluidic channel into a plurality of optical filters, wherein a first of the filters is conductive of substantially all visible light components, and a second of the filters is conductive of a subset of the visible light components; communicating at least some of each of the conveyed first and second portions of the second light to a photodetector; and outputting a color-space-time signal from the photodetector.
These and other aspects and implementations are described in detail in the drawings, the detailed description and the claims.
Examples of devices, systems and techniques for flow cytometry described in this document implement various technical features that are either alone or in various combinations can be used to achieve one or more technical benefits. One of the technical features, for example, is to provide optical illumination to the fluid in a flow cytometer device or system to allow optical sensing or monitoring in performing flow cytometry operations. The functionality of microfluidics can be combined with photonics to create a technology platform that provides integrated microfluidic photonics. Embracing photonics is a logical path of evolution for microfluidics, as a wide range of techniques for biological and chemical detection are photonic in nature. Fluorescence, fluorescence resonance energy transfer (FRET), optical scattering, and surface-enhanced Raman spectroscopy (SERS) are examples of effective and accurate methods to detect analytes at the cellular and molecular level. Integration of microfluidics with photonics represents not only a new technology platform but also a transformation to the new paradigm of bio-system-on-a-chip (BSoC). Integrated microfluidic photonic circuits have promising applications in biomedicine.
A flow cytometer based on FACS (fluorescence-activated cell sorter) is one example for combining photonics and microfluidics to meet some of the aforementioned requirements and can be a valuable bioanalysis tool for characterizing physical and biochemical properties of various chemical or biological particles, e.g., molecule clusters and cells, in a highly quantitative manner, and for detecting and monitoring the progression of diseases such as acute myeloid leukemia (AML) and AIDS. With the addition of the cell sorting capability to enrich the purity of biospecimens and extract rare cell types, a FACS can interrogate and sort cells with a throughput of tens of thousands of cells per second, making possible rare-event studies such as identification of bacterial cells or isolation of stem cells or tumor cells.
Various technical features described herein can be used to form a flow cytometer based on FACS. With the advent of lab-on-a-chip technologies, bulk optics in a FACS can now be replaced with integrated optics, affording some level of device miniaturization and cost reduction. The availability of small and inexpensive diode lasers—originally developed for optical disk and other devices—has also provided impetus for the development of micro FACS. Multicolor detection that scales with a lab-on-a-chip platform can be achieved in an integrated system described in this document by eliminating multiple photodetectors such as PMTs and by providing signal encoding in different optical signals generated in the system.
The actuator for sorting particles in
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In
In the specific example in
The system in
In
One of technical limitations in some other flow cytometry systems is using multiple PMTs to respectively detect optical signals at different fluorescent wavelengths. Presence of multiple PMTs in such systems complicates the system design, increases the cost, and renders the systems bulky and heavy. One of technical features described in this document is to provide signal encoding in multiple different optical signals so that different optical signals are encoded with unique and mutually different or orthogonal codes. As such, these optical signals can be multiplexed together for optical detection by a single optical detector and the information carried by the different optical signals can be separated by demultiplexing based on the unique and mutually different or orthogonal codes. The demultiplexing can be performed via digital signal processing.
In
In
Hence, the above examples for optical encoding and decoding in flow cytometry are based on an optical interrogation method with a single optical detector based on signal encoding via optical signal structures like optical encoders illustrated in
Specific examples of signal encoding and decoding based on a COlor-Space-Time (COST) encoding are described below in which an improved flow cytometry system can be achieved by COST to support detection of multiple (e.g., 20 or more) fluorescent wavelengths using a single detector and, more particularly in at least some embodiments, a single photo-multiplier tube (PMT) or single-photon avalanche detector (SPAD) or avalanche photodiode. In at least some embodiments, the improved flow cytometry system is implemented using lab-on-a-chip technology and architecture. A simpler version of such architecture (which can be referred to as space-time coding) in at least some embodiments is also provided to allow for multi-point detection and the consequent generation of “verification signals” to record sorting efficiency and accuracy in real time.
The following examples include methods and architectures, and/or devices embodying such, for COST coded detection of multiple fluorescent wavelengths using a single detector within a lab-on-a-chip fluorescence-activated cell sorter (FACS) or flow cytometer. Such embodiments can be considered an extension of space-time coding, which is modified to include color coding by incorporating color dyes in the waveguides transmitting the fluorescence to the detector. With the appropriate choice of dyes and calibration of the absorption spectrum, twenty or more fluorescent wavelengths can pass through the color-filter waveguides and be detected using a single detector such as a PMT or SPAD. Although in some embodiments of the present invention, colored waveguides/filters are integrated on a chip to achieve COST coded detection, in other embodiments it is also possible to implement the COST concept using one or more external color filters not integrated with the chip. In such case, when the chip is disposed of after a single use or a few uses, the color filter(s) is/are not (or need not be) disposed of
In at least some additional embodiments, the flow cytometer and/or FACS also include one or more additional components and/or features. These can include, for example, an array of integrated lenses that focus light and shorten the interrogation zone to enhance detection throughput. Also, these features can include flow disturbance minimization, 3D flow confinement and/or cascaded sorting strategies to achieve >1M enrichment factor with minimum cell loss. Also, these features can include system integration architectures with real-time electronic control and signal processing algorithms to coordinate detection and sorting, enhance sensitivity and minimize sorting error. In at least some embodiments, the COST approach provides an integrated, optofluidic solution to multicolor detection thus enabling the construction of FACS or flow cytometers that are orders of magnitude smaller, lighter and/or less expensive than existing commercial systems.
In at least one embodiment, a system for flow cytometry includes a microfluidic channel, and a first light conveying structure configured to convey substantially all visible light components, and having a first end proximate the microfluidic channel. The system also includes at least one second light conveying structure having at least one second end proximate the microfluidic channel, and extending substantially alongside the first light conveying structure, where the at least one second light conveying structure is configured to convey at least one subset of the visible light components. The system further includes a light sensing device arranged proximate respective additional ends of each of the light conveying structures, the respective additional ends being respectively opposite the respective first and second ends. Respective portions of light emanating from material passing through the microfluidic channel are received by the respective light conveying structures and communicated at least in part thereby to the light sensing device, whereby an indication of the material passing through the microfluidic channel can be determined based upon one or more signals output by the light sensing device.
Additionally, in at least one embodiment, a method of performing flow cytometry includes injecting first light into a microfluidic channel through which material is passing, and receiving second light from the microfluidic channel into a plurality of waveguides, where a first of the waveguides is conductive of substantially all visible light components, and a second of the waveguides is conductive of a subset of the visible light components. The method further includes conveying first and second portions of the second light through the first and second waveguides from respective first ends of the waveguides to respective second ends of the waveguides, communicating at least some of each of the conveyed first and second portions of the second light to a photodetector, and outputting a color-space-time signal from the photodetector.
Further, in at least one embodiment, a method of performing flow cytometry includes injecting first light into a microfluidic channel through which material is passing, and receiving second light from the microfluidic channel into a plurality of optical filters, where a first of the filters is conductive of substantially all visible light components, and a second of the filters is conductive of a subset of the visible light components. The method additionally includes communicating at least some of each of the conveyed first and second portions of the second light to a photodetector; and outputting a color-space-time signal from the photodetector.
Referring to
In the embodiment in
The laser light source 4 in the present embodiment takes the form of a 405/488 nm (or Blu-ray standard) laser. In other embodiments, a variety of other excitation lasers can be used instead (e.g, lasers at 630-650 nm and/or other lasers manufactured by a variety of to companies such as Nichia, Sony, Xerox, Omicron, etc.). Additionally, in the embodiment of
Turning to
In the present embodiment, to improve reliability and reusability of the FACS device 10 as a lab-on-a-chip device, the microfluidic channel 20 is made of polydimethylsiloxane (“PDMS”) and, additionally, the PDMS surfaces that are in contact with fluid (e.g., the interior surfaces of the channel) are further coated with a thin, smooth, uniform layer of amorphous Teflon (Teflon AF), particularly a Teflon coating having a lower refractive index (e.g., ˜1.31) than that of water (˜1.33). Use of the Teflon coating alleviates concerns (which can be present with a variety of PDMS-based microfluidic devices) associated with the porosity and permeation properties of PDMS (which can present concerns especially when dealing with small molecules).
In addition to the above benefits, another benefit of employing the Teflon-coated microfluidic channel 20 is that it facilitates the operation of the microfluidic channel additionally as a low-loss optical waveguide. That is, through the use of the Teflon-coated microfluidic channel 20, in the present embodiment light 46 entering the FACS device 10 from the multimode fiber 6 during operation generally is directed into and guided within the microfludic channel 20 (as indicated by an arrow 48) toward and into the sampling region 40 of the microfluidic channel 20. Upon reaching the sampling region 40, the light 46 impinges the cells or other matter of interest passing through the microfluidic channel and causes fluorescent light to be emitted, some or all of which the enters into the additional waveguide structure 50 arranged along a side 44 of the microfluidic channel 20/sampling region 40.
The above-described implementation of lab-on-a-chip technology is particularly advantageous insofar it constitutes an architecture that allows for multiple detection points along the flow path to enhance sensitivity and suppress noise. In some conventional flow cytometry device architectures, light from a light source (such as an excitation laser source) suffers from power splitting loss. In other words, if a cell (or other subject matter of interest) passes several (e.g., 4) different optical interrogation zones, the excitation laser power may be divided at each of those zones (e.g., divided 4 times) in a manner that results in excessive splitting loss (e.g., 6 dB splitting loss). In contrast, using the above-described embodiment employing the Teflon-coated microfluidic channel 20, the channel conducting the cells (or other subject matter of interest) serves also as the excitation light-guiding waveguide, and consequently it is possible to achieve multi-point optical interrogation as discussed further below. At the same time, the optical intensity of the guided light is lower than a tightly focused laser beam spot to avoid the effect of photo bleaching.
Referring additionally to
Each of the first, second, third and fourth waveguides 52, 54, 56 and 58 has a different respective color. More particularly, the first waveguide 52 is transparent with no particular color (e.g., clear), while the second waveguide 54, third waveguide 56 and fourth waveguide 58 are red, green and blue, respectively. Consequently, while the first waveguide 52 is able to transmit all (or substantially all) components of light within the visible light spectrum (e.g., all light components having wavelengths within the range of about 380 nm to 750 nm, or “white light”), the other waveguides 54, 56, and 58 tend to only transmit red, green and blue light components, respectively, with other colored light components being partially filtered out. Thus, the waveguides 54-58 can also be considered optical filters. As for the first filter structure 60, this structure encompasses several block features 66 that are black or blackened/darkened and that limit the ability of light to proceed form the sampling region 40 to the waveguides 52-58. Further, with respect to the second filter structure 64, this structure also includes block features 68 that are blackened/darkened and that restrict the ability of light to proceed out of the waveguides 52-58 and out of the FACS device 10 toward the PMT 14. The features 66, 68 in particular serve to increase the contrast ratio and reduce crosstalk, and further serve as a beam block for optical isolation.
Typically, it is desirable to take care with optimizing and characterizing the various waveguides 52-58 (and particularly 54-58) to obtain desired operation. To create the red, green and blue waveguides 54-58 as well as the filter structures 60, 64, red, green, blue, and black color dyes are respectively injected into the transverse waveguides and the filter structures. In the present embodiment, the color dyes are oil soluble and can be mixed with high-index (e.g., n=1.42 to 1.46) PDMS to form a colored optical waveguide/filter structures. These high index PDMS prepolymers fill the waveguide channels, which are formed using low-index (n=1.41) PDMS. By properly choosing the color dye or a mixture of different dyes and by calibrating the absorption spectrum, the waveguides 54-58 can each have a respective desired transmission spectrum. In the present embodiment, to cover the maximum number of wavelengths, the center wavelength for the three color filters should occur at around 510 nm, 570 nm, and 640 nm.
In some implementations, each of the red, green and blue (RGB) waveguides (which as mentioned above also can be considered optical filters) 54-58 can be designed to exhibit a gradual change (rather than rapid cutoff) in its transmission characteristics with wavelength. If a single dye is not able to produce the desired spectral response, mixture of dyes may be used. Further, by appropriately coloring/darkening the features 66, 68 of the filter structures 60, 64, and appropriately choosing the shapes and arrangements of those features, light can be appropriately directed from the sampling region 40 to the waveguides 52-58 as well as directed out of the waveguides 52-58 toward the PMT 14. After the color filter design is chosen, optical design software such as ZEMAX (as available from Zemax Development Corporation of Belleview, Washington) can be used to further design the COST coding FACS system 2.
Referring still to
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It should be understood that, depending upon the embodiment, the additional waveguide structure 50 can be formed by multiple layers of materials. Referring additionally to
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The three waveguides 122 of the waveguide array each conduct light away from the microfluidic channel 126 as indicated by an arrow 128, and provide their light output to a single PMT detector. Consequently, as one of the cells 118 travels along the microfluidic channel 126 successively past the waveguides 122 of the waveguide array, three serial peaks separated by the time of travel are detected via the PMT, thus converting the space signal (representing the cell positions) into a space-time coded output signal or time-domain signal 130. Using a digital match filter to match the waveform of the time-domain signal, one can suppress noise and obtain the travel speed of each individual cell, thus establishing the timing for downstream sorting. Further, if one chooses to interrogate a given one of the cells 118 multiple times along its path (e.g. oversampling to enhance the signal-to-noise ratio or to verify whether the cell is sorted to the right channel), it is possible to alter the coding patterns so that the resulting time-domain signal at each point of detection can be extracted using a corresponding match filter.
Turning to
Although the waveguide 52 was described above as being a single clear waveguide, as with the array of waveguides 122 shown in
The above-described COST technology offers significant benefits in system to functionality and cost. Further, assuming particular design constraints, the technology also is consistent with satisfactory device throughput. In particular, assuming the entire transverse waveguide area takes 100 um (in width) and the cell travels at 50 cm/s, the time to pass the optical interrogation zone (that is the zone defined by the outermost edges of the outermost waveguides 52, 58 of the additional waveguide structure 50) within the sampling region 40 is 0.2 ms. This limits the detection throughput to 2,000 to 5,000 cells/s or in the order of 10M cells/hr. Although this can be a satisfactory number for some applications, it still falls short in certain other applications. Therefore, to further increase the throughput, it is further proposed that in certain embodiments in-plane lenses to implement the COST design. In such an integrated lens approach, a lens array creates a series of focal spots that are separated by less than 5 um from each other, thus reducing the total width of the interrogation zone to be around 25 microns. As a result, the time to travel through the COST region becomes less than 50 us. This design can potentially increase the throughput to 20-30 K/s or about 100M cell per hour.
Depending upon the embodiment, additional structures can be used to further enhance performance of the FACS and/or other flow cytometry designs described herein. For example, in at least some embodiments, prisms and other structures can be used as described for example, in U.S. patent application Ser. No. 12/152,665 filed on May 14, 2008 entitled “System and Method for Flow Cytometry”, U.S. provisional patent application 61/068,198 filed on Mar. 5, 2008 also entitled “System and Method for Flow Cytometry”, and further U.S. provisional patent application 60/917,848 filed on May 14, 2007 and entitled “Light Conveying Device”, each of which is hereby incorporated by reference herein.
In
The multi-parameter on-chip detection and the cell sorting need to function in a well coordinated manner controlled by a real-time electronic system. Sensitivity, latency, and timing jitter are three key issues a good electronic control system needs to address. Sensitivity depends on the quality of the device itself and on the effectiveness of the real-time signal processing capability embedded in the electronic system. Latency is the amount of time required for the algorithms to complete computation. Timing jitter is the variation in latency. The control circuit architecture can be implemented in: (1) analog circuits, (2) microprocessors, or (3) application specific integrated circuit (ASIC). Because of the difficulty in implementing advanced signal processing algorithms in analog circuits and the limited computational power of microprocessors that yields long latency and large timing jitter, the ASIC approach may be implemented for the control circuit. For example, the National Instruments compactRIO system provides a complete embedded system with real-time operating system (RTOS) running on a microprocessor and a field-programmable-gate-array (FPGA), which is basically a highly cost-effective type of ASIC. This system may be used for the control.
The RTOS provides a device driver to access the Ethernet connection chips and the TCP/IP protocol stack for internet communication. This connection is important for data feedback from the compactRIO system and for controlling the real-time hardware. The real-time algorithm can be implemented in the FPGA and the timing jitter is expected to be less than 10 us. In the proposed approach, the electronic control provides the following 3 functions: (1) increasing signal-to-noise ratio (SNR) to improve detection efficiency, (2) instant cell speed estimation to improve sorting accuracy, and (3) sorting signal generation through a waveform generator.
The real-time processing control unit shown in
To increase the accuracy of cell detection, three forms of noise that affect the sensitivity can be considered and addressed in designing the detection circuitry: (1) thermal noise of the detection circuit that is nearly white Gaussian noise (WGN), (2) PMT or SPAD dark count noise, and (3) low frequency noise due to laser power fluctuation and stray light. Understanding the characteristics of the noise spectrum, signals can be generated using the aforementioned space-time and COST design so that the signal frequency band has the least overlap with the noise spectrum. Under the WGN condition, the highest S/N ratio can be achieved with the design of match filter, a filter having a response that is the inverse reciprocal of the waveform of the signal. The finite impulse response (FIR) implementation of the match filters is illustrated in
In some implementation, real-time cell speed estimation can be implemented for high accuracy single-cell sorting. As the speed of the flowing cells changes, the signal generated from the passing cell changes as well. If the cell speed in the microfluidic channel increases, the signal duration becomes shorter. If the speed of each cell varies in a random fashion, the variation of cell speed can be treated as an additional source of noise. It affects both the S/N ratio and the timing jitter. A more effective match filter can be designed based on the knowledge of the speed of each cell and the match filter can be programmed accordingly. The acquired information of cell speed can also be used to adjust the timing control for high accuracy single-cell sorting.
In the frequency domain analysis, variations in cell speed can be treated as variations of the frequency response of the signal. An increase in cell speed adds more high frequency components to the signal, as illustrated in
The signal encoding structures used in
Referring to optical filter design for COST coding in
The particle sorting mechanism in flow cytometry devices and systems can be implemented in various configurations. The following sections provide a particle sorter based on a piezoelectric actuator which can be configured to operate with low voltage (typically less than 10 Vp-p), having low power requirements (typically less than 0.1 mW), and having a fast response time of approximately 0.1-1 msec with particle flow speeds of approximately 1-10 cm/sec. The particle sorting system is operable in a closed loop manner using a spatial filter and processing techniques for determining the presence of a particle by analyzing a light signal over time, which signal is output by a detector.
Referring to
A piezoelectric actuator 118 operates to cause a flow disturbance to fluid in the actuation area 114 in response to a control signal such as a voltage control signal from a controller or driver as illustrated in
More completely,
The piezoelectric actuator is formed using a first layer 136 such as stainless steel or copper and a second layer 134 such as lead zirconate titanate. Lead zirconate titanate has a chemical formula of Pb[ZrxT11-x]O3, where 0<x<1, and is a ceramic perovskite material that shows a marked piezoelectric effect. It is also known as PZT which is an abbreviation of the chemical formula. Contact pads 38 are provided for application of the control signal across the two layers.
The piezoelectric actuator 118 is integrated with the PDMS substrate by first forming a hole in the PDMS substrate, such as by using a 16 mm diameter punch, and then both the PDMS substrate and the piezoelectric actuator 118 are UV ozone treated for another five minutes. The actuator is then aligned and brought into contract with the PDMS substrate 130, and the sorter 10 is then baked at 85 degrees C. for 8 hours.
As shown in
The above spatial filter in
Optofluidics is an emerging field that integrates microfluidics and optics on the same device to work synergistically. Devices that contain both microfluidic channels and on-chip photonic circuits, such as integrated biochemical sensors, show enhanced functionality and sensitivity and enable significant cost and size reduction. In order to assure that photons and biological samples in the fluid interact most effectively for the highest sensitivity, however, we desire the flexibility to direct and align the paths of light and fluid. In some cases, we need light beams to intersect the fluidic channels to localize the interrogation area. In other cases, we want the light wave and the fluid to share the same path to maximize their interaction. For the latter case, we still lack an effective fabrication method. Due to the fact that most polymers used in lab-on-a-chip devices have a higher index of refraction than water, light traveling in the fluidic channel will not be confined, suffering from high radiation loss. A PDMS-compatible process is provided here for coating microfluidic channels with a layer of low refractive index Teflon AF solution, enabling the water in the fluidic channel to be used as the waveguide's high index core. The Teflon AF coated waveguide works not only for straight fluidic channels but also for split channels. In addition to delivering the light, by Teflon coating the microfluidic channel, a channel is created with low sample adsorption, avoiding a troublesome problem found in many polymer-based microfluidic devices.
Teflon AF is an amorphous fluoropolymer that is chemically stable and optically transparent from UV to IR wavelengths . Unlike other fluoropolymers, Teflon AF has a refractive index (n=1.31) that is lower than the index of water (n=1.33), therefore a Teflon AF coating layer can be used to clad a liquid-core optical waveguide. Light will then be delivered through the same physical path as the fluid flows by total internal reflection (TIR) when the coated channels are filled with water or aqueous solutions. A procedure for coating Teflon AF onto PDMS channel walls is provided here by flowing Teflon AF solution through the micro channel, thereby creating the cladding layer for an optical waveguide along the path of fluid flow. The light introduced to micro channels is confined inside the core of the waveguide (i.e. microfluidic channel) and guided by fluid flowing through the channel.
Microfluidic channels that are 200 μm by 70 μm are fabricated in PDMS. A master mold is lithographically defined on an optically smooth Si wafer using SU-8 50 (MicroChem). Two replicas are created: one replica with microfluidic channels and one replica of an optically smooth blank Si wafer. A solution of 2% 1H,1H,2H,2H-perfluorodecyltriethoxysilane (Sigma Aldrich Inc.) is spin-coated onto PDMS substrates and heated at 110 oC for 10 minutes to promote adhesion between PDMS and the Teflon AF solution. Both PDMS surfaces are then activated for permanent bonding by UV/Ozone treatment (UVO-CLEANER 42, Jelight Inc.) for 3 minutes and bonded together, thus capping the microfluidic channels. A 6% Teflon AF solution (601-S2, DuPont Corp) is flowed into the microfluidic channels. Once they are filled, vacuum (P=-20kPa) is applied for 20 minutes to remove excess Teflon AF solution from the channels (see
The process results in a smooth channel with a hollow core. The Teflon AF-coated PDMS device is heated to 155 oC for 20 minutes to evaporate the fluoroinert solvent, and then heated further to 175 oC (15 oC above the its glass transition temperature) for 20 minutes to form a smooth Teflon AF layer. This relatively low temperature coating is compatible with PDMS process while significantly reducing the consumption of Teflon solution compared to the spin-coating process. Calculations show that a ˜5 μm thick Teflon AF film is necessary to confine the light to the liquid core. In some implementations, the cladding thickness is typically 5 to 15 μm, thick enough to confine and guide light waves. The thickness of the Teflon AF coating layer can be further controlled by adjusting the applied vacuum pressure and concentration of the Teflon AF solution. After slowly cooling the devices to avoid cracking due to thermal mismatch, an optical fiber is inserted into the channel for light coupling. Deionized (DI) water is then introduced into the hollow core to serve as both the sample flow carrier and the core of the optofluidic waveguide.
The flowing DI water transports both the suspended samples and the light in the same channel.
As shown in
While this document contains many specifics, these should not be construed as limitations on the scope of an invention or of what may be claimed, but rather as descriptions of features specific to particular embodiments of the invention. Certain features that are described in this document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or a variation of a subcombination.
Only a few implementations are disclosed. However, variations and enhancements of the described implementations and other implementations can be made based on what is described and illustrated in this document.
Claims
1-28. (canceled)
29. A microfluidic particle sorting device comprising:
- a microfluidic input channel for receiving a sample fluid, the sample fluid comprising a plurality of particles;
- a particle sorting junction coupled to the microfluidic input channel;
- a plurality of microfluidic output channels in fluid communication with the sorting junction, the plurality of output channels comprising a first output channel and a second output channel; and
- a piezoelectric actuator in fluid communication with the sorting junction that bends in a first direction in response to a first sorting control signal to direct a target particle of the plurality of particles to the first output channel and bends in a second direction in response to a second sorting control signal to direct the target particle to the second output channel.
30. The microfluidic particle sorting device of claim 29, wherein the plurality of microfluidic output channels further comprises a third output channel, wherein the target particle flows to the third output channel when the piezoelectric actuator does not deflect.
31. The microfluidic particle sorting device of claim 29, wherein the first sorting control signal comprises a positive voltage and the second sorting control signal comprises a negative voltage.
32. The microfluidic particle sorting device of claim 29, further comprising an actuation chamber, wherein the piezoelectric actuator is coupled to the actuation chamber, and wherein the piezoelectric actuator is in fluid communication with the sorting junction through the actuation chamber.
33. The microfluidic particle sorting device of claim 32, wherein the piezoelectric actuator bends in the first direction towards the actuation chamber and bends in the second direction away from the actuation chamber.
34. The microfluidic particle sorting device of claim 29, wherein the piezoelectric actuator bending in the first direction causes a flow disturbance in a first flow disturbance direction that directs the target particle to the first output channel and bending in the second direction causes a flow disturbance in a second flow disturbance direction that directs the target particle to the second output channel.
35. The microfluidic particle sorting device of claim 29, wherein the piezoelectric actuator comprises a layer comprising lead zirconate titanate.
36. The microfluidic particle sorting device of claim 29, wherein the plurality of particles comprises cells.
37. The microfluidic particle sorting device of claim 41, wherein the plurality of particles comprises beads.
38. A microfluidic particle sorting system comprising:
- an optical detector; and
- a microfluidic cartridge comprising: a microfluidic input channel for receiving a sample fluid, the sample fluid comprising a plurality of particles; a particle sorting junction coupled to the microfluidic input channel; a plurality of microfluidic output channels in fluid communication with the sorting junction, the plurality of output channels comprising a first output channel and a second output channel; and a piezoelectric actuator in fluid communication with the sorting junction that bends in a first direction in response to a first sorting control signal to direct a target particle of the plurality of particles to the first output channel and bends in a second direction in response to a second sorting control signal to direct the target particle to the second output channel.
39. The microfluidic particle sorting system of claim 38, where the optical detector is coupled to the first output channel to detect a presence of the target particle in the first output channel.
40. The microfluidic particle sorting system of claim 39, wherein the optical detector produces a branch verification signal upon detecting the presence of the particle in the first output channel.
41. The microfluidic particle sorting system of claim 40, further comprising a particle sorter control module in communication with the optical detector, wherein the particle sorter control module receives the branch verification signal from the optical detector and verifies whether the target particle is directed into the first output channel.
42. The microfluidic particle sorting system of claim 41, wherein the particle sorter control module further comprises a delay counter that delays actuation of the piezoelectric actuator for an amount of time.
43. The microfluidic particle sorting system of claim 42, wherein the amount of time equals a travel time of the target particle arriving at the sorting junction.
44. The microfluidic particle sorting system of claim 42, wherein the amount of time is adjustable.
45. The microfluidic particle sorting system of claim 41, wherein the particle sorter control module records a sorting efficiency.
46. The microfluidic particle sorting system of claim 38, wherein the optical detector detects an optical signal carrying information indicative of a property of the target particle.
47. The microfluidic particle sorting system of claim 46, wherein the optical signal comprises light emitted or scattered from the target particle when the target particle is in the input channel.
48. The microfluidic particle sorting system of claim 38, wherein the optical detector comprises a photomultiplier tube.
Type: Application
Filed: Jun 7, 2019
Publication Date: Feb 27, 2020
Applicant: The Regents of the University of California (Oakland, CA)
Inventors: Yu-Hwa Lo (San Diego, CA), Chun Hao Randy Chen (Arcadia, CA), Sung Hwan Cho (La Jolla, CA), Shang-Feng Tsai (Sunnyvale, CA)
Application Number: 16/435,354