The intersection of flow cytometry with microfluidics and microfabrication

Abstract

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.

Fig. 1

A schematic of a microfluidic flow cytometer constructed in 1965 by Kamentsky et. al. and used to analyse cells at 1000 cells/s. The bowtie channel shown was ultrasonically cut in a cover slip. The narrow section between the inlet and outlet was 100 μm deep and 100 μm wide. From [L. A. Kamentsky, M. R. Melamed and H. Derman, Science, 1965, 150, 630–631]. Reprinted with permission from AAAS.

Fig. 2

A common flow cytometer analysis configuration. (A) shows an example using a pressure differential that allows for sample to be delivered slowly into a fast moving sheath stream. The laser is shaped and focused to the flow cell using crossed cylindrical lenses onto the flow cell. The dashed square is magnified in panel B, but the collected light is directed through a set of dichroic mirrors and filters to photodetectors that are connected to the data acquisition system. (B) A magnified view that shows the hydrodynamic focusing cone where the narrow diameter sample tube is surrounded by the fast moving sheath fluid. This results in hydrodynamic focusing of the sample stream as it passes through the flow cell. The focused light strikes a blocker bar in front of the forward scatter detector, which only allows low angle scattered light to reach the detector. (C) 90° light is collected via high numerical collection optics, which is often accomplished via a gel coupled lens. The hydrodynamic focusing ensures that the particle passes through the interrogation volume in the centre of the flow profile. (D) The interrogation volume is made up of the intersection of the laser with the flow stream, while the larger analysis volume is defined by the field of view of the collection optics.

Fig. 3

A common flow sorting flow cell configuration. It is also possible to perform analysis directly in the jet as it exits the focusing region.

Fig. 4

Online sample preparation of blood cells. (A) The CTC-iChip. From [E. Ozkumur, A. M. Shah, J. C. Ciciliano, et. al. Science translational medicine, 2013, 5, 179ra147] Reprinted with permission from AAAS. (B) Acoustophoretic separation of cancer cells from blood using in an etched silicon microchannel. Figure reproduced from [P. Augustsson, C. Magnusson, M. Nordin, H. Lilja and T. Laurell, Analytical chemistry, 2012, 84, 7954–7962] with permission of the American Chemical Society Copyright 2012.

Fig. 5

High throughput and high content screening systems for flow cytometry. (A) An image of samples separated by air bubbles in a delivery line in a sampling system for high throughput screening via flow cytometry. Figure reproduced from reference with permission from John Wiley & Sons, Inc. (B) A system that uses 384 parallel microchannels and a laser scanning detection system to provide images of cells during high content screening. Reprinted by permission from Macmillan Publishers Ltd: [Nature Methods] (B. K. McKenna, J. G. Evans, M. C. Cheung and D. J. Ehrlich, Nature methods, 2011, 8, 401–403), copyright (2011) (C) A system that generates a library of droplets, stores them in a delay line, mixes them with a reagent, and then performs flow based optical analysis. Reproduced from Ref. with permission from The Royal Society of Chemistry.

Fig. 6

Sheathless microfluidic focusing techniques. (A). Bulk acoustic focusing in microfabricated silicon devices. A common input is split into many channels and an applied frequency is tuned to generate single focused stream in each channel. Reprinted from Methods, v. 57, P. P. Austin Suthanthiraraj, M. E. Piyasena, T. A. Woods, M. A. Naivar, G. P. Lopez and S. W. Graves, One-dimensional acoustic standing waves in rectangular channels for flow cytometry, p. 259–71. Copyright (2012), with permission from Elsevier. (B,C). Surface acoustic focusing in PDMS microchannels. (B) Surface acoustic waves are generated via integrated IDTs fabricated on a piezo substrate that focus 1.9 μm green microspheres into a pressure node. (C) As the microspheres flow from left to right they are acoustically focused into the central stream. The focusing is shown at four points, where the top most image is taken from square I, the next lower from II, the next from III, and the bottom most image from square IV. Figures (B,C) reproduced from reference with permission from The Royal Society of Chemistry. (D,E) Inertial focusing of particles/cells in a highly parallel PDMS microchannel system. (D) Randomly distributed particles/cells introduced from the inlet and inertially focused based on the size, along the length of the channel. (E) A micrograph of inertially focused particles. Figures (D,E) reproduced from reference with permission from The Royal Society of Chemistry. (F, G) Dielectrophoretic particle focusing. (F) A schematic of microelectrodes integrated with the fluidic channels. (G) 6μm particles are focused in the presence of an electric field. Figures (F,G) were reprinted from Biosensors and Bioelectronics, Vol 21, D. Holmes, H. Morgan and N. G. Green, High throughput particle analysis: Combining dielectrophoretic particle focussing with confocal optical detection, Pages 1621–1630, Copyright (2006), with permission from Elsevier.

Fig. 7

Examples of optical detection and analysis of typical flow cytometry application areas. (A) The typing of white blood cells by a combination of side scatter and detection of CD45 and CD4 cell surface markers. In the top panel, the leucocyte population is selected for via an electronic gate (the larger rectangle on the bivariate plot of side scatter vs. anti-CD45 generated fluorescence). In the lower panel the resultant CD4+ lymphocyte population is counted via a rectangular electronic gate. The smaller rectangle in the upper panel can be used to count the total lymphocyte population in the sample. This value can be used to provide a percentage of CD4+ lymphocytes, which is useful for paediatric cases of HIV (122). Figure reproduced from reference with permission from John Wiley & sons. Inc. (B) An extensive immunological panel where many markers are being identified simultaneously for cell typing. Up to seventeen colours are used to identify many markers. This approach uses cluster analysis to identify cell populations, which are shown in different colours. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology] (reference 21), copyright (2004) (C) Each population of a 64-plex microsphere set resolved by their response in two colours of fluorescence. The populations can be gated on as shown for cells in panel A and the reporting fluorescence for the assay on the microsphere can be discriminated from the overall population. Figure reproduced from reference with permission from John Wiley & Sons, Inc.

Fig. 8

Commercial microfluidic systems. (A) The Agilent 2100 Bioanalyzer cell analysis chip The letter B denotes where the buffer is input. The S denotes the sample input. Whereas the P and D denote the priming well and reference dye well respectively. The particles from the sample are delivered in a microchannel (left hand circle), focused against the channel wall by sheath fluid (center channel), and analysed using a red diode laser or blue LED for excitation (right hand circle). Figure reproduced from reference with permission from John Wiley and Sons, Inc. (B) The Fishman-R microfluidic flow cytometer. Figure reproduced from reference with permission from John Wiley and Sons, Inc.