Flow cytometry has evolved from its initial purpose as a cell counting technique into a powerful and essential phenotypic characterization platform. The recent addition of acoustic focusing, and principles of microfluidic and nanotechnology devices, have allowed investigators to extend the capabilities of cytometry to high-throughput screening, and the collection and study of subcellular compartments and rare cell types.
The foundation of flow cytometry resides in the principle that electrical current applied to particles moving through an aperture results in impedance proportional to their volume. Wallace Coulter patented this concept and applied it to his eponymous cell counter. In the 1970s, the Los Alamos laboratories expanded upon the Coulter Principle and generated devices in which laser light scattered by cells passing through an interrogation window could be used to infer parameters such as size, membrane integrity, and proliferation. The experimental generation of antibodies specific to cell-surface proteins, and the more recent creation of elegant genetic reporter systems, have facilitated characterization of different cell types via excitation of conjugated fluorophore compounds or naturally fluorescent fusion proteins by corresponding laser wavelengths.
Through these innovations, it is possible to identify, quantify, and sometimes collect and subculture important cell types from otherwise heterogeneous populations. These are capabilities for which venerable molecular and biochemical techniques such as qPCR and western blotting are inadequate, as they largely address samples as static lump sums without internal variation. With increasingly powerful flow platforms, investigators are within reach of objectives such as improving efficiency and viability in stem cell-based strategies to derive therapeutically relevant cell types.
Conventional flow cytometry
Conventional flow cytometry uses hydrodynamic focusing to align cells into single-file arrangements so that they pass the interrogation point as individual events. Sheath fluid within a long tube acts as the conduit into which the instrument injects and focuses the sample to create a core stream. Within the sheath, hydrodynamic forces overtake investigator control, and particles rapidly accelerate, such that adherence to Poisson distribution limitations places a restrictive cap on events detected and recorded, and sample volume injected into the stream. The accurate detection limits of conventional flow cytometry vary, but hover around 25,000 cells per second, with diameters between about one and 20 microns, often in volumes of a milliliter or less, in the interest of optimizing assay duration and efficiency. When exceeded, instrumentation can clog, or record too many doublet events for data to be informative, or simply perform inadequately to register the desired number of events in the time allotted before the next user loses his patience. These limits are refractory to the modern demands of cell biology, which place a premium on throughput, and on the identification of rare cell types, esoteric, or novel events. Adherence to these goals can often necessitate concentration of media through time-consuming centrifugation steps. Moreover, the small volumes required in the interest of machine run-times translate to high percentages lost in void volumes, their contents squandered prior to injection and after post-run expulsion, frustratingly lost in the final analysis.
Acoustic-assisted focusing and increased throughput
The recent addition of acoustic focusing to conventional flow cytometry goes a long way toward improving throughput and optimizing identification and collection of rare events. Acoustic-assisted hydrodynamic focusing is an evolving platform that can focus a broader array of particle sizes, more precisely, and in wider channels, than in conventional instruments. Therefore, it employs longer transit times with reduced linear velocity, enabling a higher rate of analysis, with sample volumes up to an order of magnitude greater than with hydrodynamic focusing alone. Increasing sample volumes comparatively minimizes void volumes and eliminates the need for concentration steps. The net result is greater efficiency and throughput, validating it as a robust technology for chemical and antibody screening.
This concept is incredibly important, considering the high barriers of cost, training, and coordination associated with use of screening core facilities, compared to retention of a benchtop machine in an individual laboratory space. One can also obtain significant populations of rare circulating tumor or endothelial cells from dilute solutions, without concentration steps that may be time consuming and deleterious to the cells themselves before analysis. Rare cells are defined as occupying less than .01 percent of the total population, so that obtaining 1,000 cells of interest would require a sample preparation containing at least 10 million total. Acoustic-assisted cytometers can efficiently handle the corresponding volumes, especially across multiple experimental conditions, while conventional instruments cannot.
Individual laboratories have begun to extend the capabilities of acoustic focusing through parallel flows and with microfluidic channels. The use of acoustic standing waves across multiple parallel streams can increase flow rates more than 50-fold, and analysis rates to over 100,000 events per second. Alternatively, traveling acoustic waves applied to microfluidic channels can simultaneously improve efficiency and decrease footprint by eliminating sheath altogether. There are benefits and disadvantages to this approach, because while the sheath spans the breadth of a conventional instrument, and intrinsically dilutes the sample flow, it also keeps cells from contacting and contaminating channel walls. Combining traveling waves with repeating curved microfluidic channel geometry can allow efficient focusing based on intrinsic physical flow forces, optimizing analytical efficiency, purity, and viability of collected cells.
Nanoscale flow and extracellular vesicles
Finally, a new and exciting field has coalesced around characterizing subcellular components, and using them as biomarkers, previously unfeasible because of the inadequate lower detection limits of conventional cytometry. This development breaks analysis of organelles and extracellular vesicles (EVs) free from laborious fixation and cryo-preparation steps, and costly electron microscopic analysis. Instruments with multiple high-powered short-wavelength lasers, and application of nanotechnology principles to detection, can accurately discern size differences in calibrated beads down to 100 nanometers, and detect liposomes half that diameter.
Consequently, NanoFlow has been used in disease modeling studies to distinguish whether cells preferably release exosomes expressing hallmark surface proteins, or comparatively smoother and larger microvesicles, which bud from cell membranes. With these and other emerging studies, investigators can move toward high-throughput analysis and staging of different cancer cells, which may have intrinsic properties that dictate the ratio of EVs they release.
The power of flow cytometry techniques to characterize subcellular compartments, and to process large volumes with high throughput, distinctly accelerates its potential as a predictive tool.