Image-Based Single Cell Sorting Automation in Droplet Microfluidics

The recent boom in single-cell omics has brought researchers one step closer to understanding the biological mechanisms associated with cell heterogeneity. Rare cells that have historically been obscured by bulk measurement techniques are being studied by single cell analysis and providing valuable insight into cell function. To support this progress, novel upstream capabilities are required for single cell preparation for analysis. Presented here is a droplet microfluidic, image-based single-cell sorting technique that is flexible and programmable. The automated system performs real-time dual-camera imaging (brightfield & fluorescent), processing, decision making and sorting verification. To demonstrate capabilities, the system was used to overcome the Poisson loading problem by sorting for droplets containing a single red blood cell with 85% purity. Furthermore, fluorescent imaging and machine learning was used to load single K562 cells amongst clusters based on their instantaneous size and circularity. The presented system aspires to replace manual cell handling techniques by translating expert knowledge into cell sorting automation via machine learning algorithms. This powerful technique finds application in the enrichment of single cells based on their micrographs for further downstream processing and analysis.

[1]  S. Nelson,et al.  A manual method for the purification of fluorescently labeled neurons from the mammalian brain , 2007, Nature Protocols.

[2]  H Korb,et al.  Magnetic activated cell sorting (MACS) — a new immunomagnetic method for megakaryocytic cell isolation: Comparison of different separation techniques , 1994, European journal of haematology.

[3]  P. Sorensen,et al.  The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner , 2018, Nature Communications.

[4]  D. Belder,et al.  Fluorescence lifetime-activated droplet sorting in microfluidic chip systems. , 2019, Lab on a chip.

[5]  Todd P. Lagus,et al.  High-throughput co-encapsulation of self-ordered cell trains: cell pair interactions in microdroplets , 2013 .

[6]  Y. Peter,et al.  On-chip refractive index cytometry for whole-cell deformability discrimination. , 2019, Lab on a chip.

[7]  Gabriel P López,et al.  Translating microfluidics: Cell separation technologies and their barriers to commercialization , 2017, Cytometry. Part B, Clinical cytometry.

[8]  A. deMello,et al.  The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. , 2015, Lab on a chip.

[9]  Roland Zengerle,et al.  Technologies for Single-Cell Isolation , 2015, International journal of molecular sciences.

[10]  Robert E. Nordon,et al.  Droplet-based single cell RNAseq tools: a practical guide. , 2019, Lab on a chip.

[11]  Chang Lu,et al.  Droplet sorting based on the number of encapsulated particles using a solenoid valve. , 2013, Lab on a chip.

[12]  P. H. Yap,et al.  Droplet optofluidic imaging for λ-bacteriophage detection via co-culture with host cell Escherichia coli. , 2014, Lab on a chip.

[13]  Byron F. Brehm-Stecher,et al.  Single-Cell Microbiology: Tools, Technologies, and Applications , 2004, Microbiology and Molecular Biology Reviews.

[14]  Jean-Louis Viovy,et al.  Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells , 2008, Proceedings of the National Academy of Sciences.

[15]  C. Lim,et al.  Microfluidic label-free selection of mesenchymal stem cell subpopulation during culture expansion extends the chondrogenic potential in vitro. , 2018, Lab on a chip.

[16]  Clemens F Kaminski,et al.  From microdroplets to microfluidics: selective emulsion separation in microfluidic devices. , 2008, Angewandte Chemie.

[17]  Yibo Zhang,et al.  Computational cytometer based on magnetically modulated coherent imaging and deep learning , 2019, Light: Science & Applications.

[18]  A. van den Berg,et al.  Label-free, high-throughput, electrical detection of cells in droplets. , 2012, The Analyst.

[19]  Unyoung Kim,et al.  Multitarget magnetic activated cell sorter , 2008, Proceedings of the National Academy of Sciences.

[20]  C. Hansen,et al.  Microfluidic single cell analysis: from promise to practice. , 2012, Current opinion in chemical biology.

[21]  Tuncay Alan,et al.  Surface acoustic wave enabled pipette on a chip. , 2017, Lab on a chip.

[22]  Sehyun Shin,et al.  Density-dependent separation of encapsulated cells in a microfluidic channel by using a standing surface acoustic wave. , 2012, Biomicrofluidics.

[23]  A. Matsko,et al.  Probing 10 μK stability and residual drifts in the cross-polarized dual-mode stabilization of single-crystal ultrahigh-Q optical resonators , 2019, Light, science & applications.

[24]  Tuncay Alan,et al.  Microfluidic plug steering using surface acoustic waves. , 2015, Lab on a chip.

[25]  Fumihito Arai,et al.  Geometrical alignment for improving cell evaluation in a microchannel with application on multiple myeloma red blood cells , 2014 .

[26]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[27]  Haakan N Joensson,et al.  Droplet size based separation by deterministic lateral displacement-separating droplets by cell--induced shrinking. , 2011, Lab on a chip.

[28]  Adam Sciambi,et al.  Accurate microfluidic sorting of droplets at 30 kHz. , 2015, Lab on a chip.

[29]  Sridhar Ramaswamy,et al.  A microfluidic device for label-free, physical capture of circulating tumor cell-clusters , 2015, Nature Methods.

[30]  R G Sweet,et al.  Fluorescence Activated Cell Sorting , 2020, Definitions.

[31]  A. Abate,et al.  Ultrahigh-throughput Mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops. , 2013, Analytical chemistry.

[32]  B. Kappes,et al.  Label-free, high-throughput detection of P. falciparum infection in sphered erythrocytes with digital holographic microscopy. , 2018, Lab on a chip.

[33]  B G Schutt.  The promise in practice. , 1967, The American journal of nursing.

[34]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[35]  Richard M Maceiczyk,et al.  Differential detection photothermal spectroscopy: towards ultra-fast and sensitive label-free detection in picoliter & femtoliter droplets. , 2017, Lab on a chip.

[36]  C. Klein,et al.  Single-cell analysis of CTCs with diagnostic precision: opportunities and challenges for personalized medicine , 2016, Expert review of molecular diagnostics.

[37]  J. Voldman.  Electrical forces for microscale cell manipulation. , 2006, Annual review of biomedical engineering.

[38]  K. Mizuguchi,et al.  Quantifying the relative immune cell activation from whole tissue/organ-derived differentially expressed gene data , 2017, Scientific Reports.

[39]  Imogen Moran,et al.  Single Cell RNA Sequencing of Rare Immune Cell Populations , 2018, Front. Immunol..

[40]  A. Ji,et al.  New cell separation technique for the isolation and analysis of cells from biological mixtures in forensic caseworks , 2011, Croatian Medical Journal.

[41]  G. Whyte,et al.  Image-based closed-loop feedback for highly mono-dispersed microdroplet production , 2017, Scientific Reports.

[42]  Stephen R Quake,et al.  Dissecting genomic diversity, one cell at a time , 2013, Nature Methods.

[43]  Han Gardeniers,et al.  Microfluidic device for DNA amplification of single cancer cells isolated from whole blood by self-seeding microwells. , 2015, Lab on a chip.

[44]  C. A. Smith,et al.  High-throughput screening of antibiotic-resistant bacteria in picodroplets. , 2016, Lab on a chip.

[45]  Ben Fabry,et al.  Microconstriction arrays for high-throughput quantitative measurements of cell mechanical properties. , 2015, Biophysical journal.

[46]  Tuncay Alan,et al.  Droplet control technologies for microfluidic high throughput screening (μHTS). , 2017, Lab on a chip.

[47]  M. Figge,et al.  Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets. , 2013, Lab on a chip.

[48]  Robert C. Wolpert,et al.  A Review of the , 1985 .

[49]  A. Abate,et al.  PCR-Activated Cell Sorting for Cultivation-Free Enrichment and Sequencing of Rare Microbes , 2015, PloS one.

[50]  Alexander B. Wiltschko,et al.  Diagnosis of iron deficiency anemia using density-based fractionation of red blood cells. , 2016, Lab on a chip.

[51]  M. Roederer,et al.  The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. , 2002, Clinical chemistry.

[52]  Frank Vollmer,et al.  Label-free optical detection of single enzyme-reactant reactions and associated conformational changes , 2017, Science Advances.

[53]  M. Elowitz,et al.  Challenges and emerging directions in single-cell analysis , 2017, Genome Biology.

[54]  Lani F. Wu,et al.  Cellular Heterogeneity: Do Differences Make a Difference? , 2010, Cell.

[55]  H. A. Pohl,et al.  Some Effects of Nonuniform Fields on Dielectrics , 1958 .

[56]  Jin Ho Jung,et al.  On-demand droplet splitting using surface acoustic waves. , 2016, Lab on a chip.

[57]  R. Nitschke,et al.  Quantum dots versus organic dyes as fluorescent labels , 2008, Nature Methods.

[58]  Tuncay Alan,et al.  Microfluidic on-demand droplet merging using surface acoustic waves. , 2014, Lab on a chip.

[59]  A. van den Berg,et al.  High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel. , 2012, Lab on a chip.

[60]  Fumihito Arai,et al.  Intelligent Image-Activated Cell Sorting , 2018, Cell.

[61]  D. Haber,et al.  Clusters of Circulating Tumor Cells: a Biophysical and Technological Perspective. , 2017, Current opinion in biomedical engineering.

[62]  H. Amini,et al.  Label-free cell separation and sorting in microfluidic systems , 2010, Analytical and bioanalytical chemistry.

[63]  A. Abate,et al.  High-throughput injection with microfluidics using picoinjectors , 2010, Proceedings of the National Academy of Sciences.

[64]  B Cox,et al.  Microfluidic sorting of microtissues. , 2012, Biomicrofluidics.

[65]  H. Bridle,et al.  Analysis of Parasitic Protozoa at the Single-cell Level using Microfluidic Impedance Cytometry , 2017, Scientific Reports.

[66]  Daniel Bratton,et al.  Development of quantitative cell-based enzyme assays in microdroplets. , 2008, Analytical chemistry.

[67]  I. Hellmann,et al.  Comparative Analysis of Single-Cell RNA Sequencing Methods , 2016, bioRxiv.

[68]  Graeme Whyte,et al.  Dynamically reconfigurable fibre optical spanner. , 2014, Lab on a chip.

[69]  Mehmet Toner,et al.  Controlled encapsulation of single-cells into monodisperse picolitre drops. , 2008, Lab on a chip.

[70]  R. Sandberg.  Entering the era of single-cell transcriptomics in biology and medicine , 2013, Nature Methods.

[71]  R. Westervelt,et al.  Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices , 2006 .

[72]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[73]  Christoph A. Merten,et al.  Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells , 2018, Cell reports.

[74]  H Bridle,et al.  Deterministic lateral displacement for particle separation: a review. , 2014, Lab on a chip.

[75]  Roberto Bellotti,et al.  Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR , 2017, Analytical and Bioanalytical Chemistry.

[76]  Paul D Adams,et al.  Pressure stabilizer for reproducible picoinjection in droplet microfluidic systems. , 2014, Lab on a chip.

[77]  Jan J. Lyczakowski,et al.  Label-Free Analysis and Sorting of Microalgae and Cyanobacteria in Microdroplets by Intrinsic Chlorophyll Fluorescence for the Identification of Fast Growing Strains. , 2016, Analytical chemistry.

[78]  F. Arai,et al.  High-speed microparticle isolation unlimited by Poisson statistics. , 2019, Lab on a chip.

[79]  Jan Paul Huissoon,et al.  An integrated microfluidic device for the sorting of yeast cells using image processing , 2018, Scientific Reports.

[80]  Sashwati Roy,et al.  Laser capture microdissection: Big data from small samples. , 2015, Histology and histopathology.

[81]  Umut A. Gurkan,et al.  Statistical Modeling of Single Target Cell Encapsulation , 2011, PloS one.

[82]  C. Trägårdh,et al.  Visualization of the drop deformation and break-up process in a high pressure homogenizer , 2005 .

[83]  F. Arai,et al.  Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering , 2019, Proceedings of the National Academy of Sciences.

[84]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[85]  Allon M. Klein,et al.  Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells , 2015, Cell.

[86]  Kenji Yasuda,et al.  An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution , 2017, Scientific Reports.

[87]  D. Holmes,et al.  Separation of blood cells with differing deformability using deterministic lateral displacement† , 2014, Interface Focus.

[88]  Dino Di Carlo,et al.  Microtechnology for Cell Manipulation and Sorting. , 2017, Anticancer research.

[89]  Olivia Freeman,et al.  Talking points personal outcomes approach: practical guide. , 2012 .

[90]  Jongyoon Han,et al.  Jetting microfluidics with size-sorting capability for single-cell protease detection. , 2015, Biosensors & bioelectronics.

[91]  David A Weitz,et al.  Beating Poisson encapsulation statistics using close-packed ordering. , 2009, Lab on a chip.

[92]  Xinjun Wan,et al.  Colour compound lenses for a portable fluorescence microscope , 2019, Light: Science & Applications.

[93]  Adel O. Sharif,et al.  Electrostatic enhancement of coalescence of water droplets in oil: a review of the current understanding , 2001 .