Flow cytometry is one of the most powerful and versatile techniques in modern biomedical science. It is the culmination of 400 years of microscopy, 150 years of dye and fluorescent chemistry, and 50 years of innovation in electronics and computer programming. Flow cytometry allows measurement and analysis of individual particles within a flowing fluid medium. For users, manipulation of this flowing stream via the fluidics system is the central point of control. For the purposes of clinical and research science, most often these particles are cells and flow cytometry can characterize cells by size, shape, cell cycle position, and survival using information gleaned from the scattering of light off their surfaces. It may also be used to immunophenotype and classify fixed or living cells based on expression of fluorescent reporters or proteins and their affinities to monoclonal antibodies, and categorize and compile events within a heterogeneous population to define several sub-populations of interest in a single sample. Therefore, unlike most molecular and biochemical assays which show mean properties across cell populations, flow cytometry identifies individual events within a sample, and can quantify the likelihood of rare events such as expression of novel cancer biomarkers. Moreover, in comparison to microscopy, flow cytometry can identify expression events with much greater efficiency and throughput (often >10,000 cells per second), with the added benefit of seamlessness between identification, analysis, and data storage that is still cumbersome even in the most impressive microscopy platforms. As a corollary, flow cytometry is adaptable to high-throughput screening (HTS) and drug discovery in ways that microscopy is not. Finally, with the addition of sorting, cells of interest can be isolated, obtained, and further manipulated downstream of data acquisition and analysis. In this way, therapeutically relevant cell types can be purified from differentiating stem and progenitor cells, and further tested in disease models.
A flow cytometer has three main functional systems: fluidics, optics, and electronics. The fluidics system manages the liquid stream from instrument input to output, injecting a sample into the flow cell at constant pressure (usually low, medium, or high) selected by the user. After the instrument is activated, the sheath fluid within achieves laminar flow, and injection of the sample in its own medium forces a core stream containing the cells to be characterized through the center of the sheath in a process called hydrodynamic focusing. Optimally functioning via Bernoulli’s principle, this results in a single-file stream of cells so that each one is detected as an individual event. Higher pressures result in faster sample transit and a wider core stream, making it more likely that the optics system will capture coincident events, adjacent cells that can obscure accurate detection and result in higher data variability between replicates. The optics system contains excitation light sources, lenses, and filters for interrogation and detection. Collision of light with sample at the interrogation point activates measurement and analysis of forward and side scatter of reflected light to indicate cell size and shape. Simultaneously, appropriate laser wavelengths excite fluorophores either expressed by cells (as in GFP reporters) or conjugated to specific antibodies with which cells have been preincubated. The electronics system handles digitization of the detector photocurrent, and analysis and storage of optical measurements.
The importance of fluidics
Of the three components, the fluidics system is the one that can be most easily altered by the end user. For starters, it is the user who executes cleaning and flushing cycles before and after experiments, and improper fluidics maintenance can lead to inconsistent data. When one prepares the flow cell by running a bleach cycle, a rushed and incomplete follow-up water cycle can lead to photobleaching of cells in the core stream, and weakened fluorescent detection. If one improperly cleans instrumentation after experiments, future flow assays can be compromised by clogging.
In addition to responsibility and diligence in maintenance, the user has dominion over the sheath fluid. The optimal sheath fluid can vary between experiments, and instruments’ fluidics systems come with recommendations for sheath compositions. The BD Accuri C6 is a benchtop cytometer that uses distilled water as sheath; it is common to add 2-phenoxyethanol as a surfactant to any sheath fluid to mitigate surface tension within the flow cell. Although phosphate buffered saline is a standard all-purpose sheath fluid, HEPES-buffered saline can be used in higher-pressure applications because it is more effective at controlling pH. These are both supplied in bulk by Leinco, and BD offers its own FACSFlow sheath fluid, a combination of sodium salts, phosphate buffers, and surfactant. Optimization of sheath fluid is a key consideration to undertake so that it does not become its own confounding variable.
As noted, one can change flow rate and thus sheath pressure to suit experimental needs. Although slow and low will inevitably result in the most accurate data collection, time can be priceless in experiments with many samples. If the fluidics system needs to stream 100 microliters per sample to count the desired number of cells, and there are 20 samples, low-speed analysis can easily take as long as four hours while high-speed analysis could be complete in under an hour. The sensitivity of the data must be weighed against the integrity of samples after sitting for so long.
Innovations in core stream focusing have improved data quality while significantly increasing flow speeds. The Thermo Fisher Attune NxT cytometer combines hydrodynamic with acoustic focusing, using ultrasound to subject the flow channel to a standing acoustic wave, moving cells to a pressure minimum. The result is a speed of > 35,000 cells/second without coincident events or sample clumping, and therefore greater adaptability to HTS or dilute samples with rare cell types. The Luminex Guava easyCyte does away with the sheath altogether and substitutes a syringe pump coupled to a microcapillary system. This design minimizes both liquid waste and footprint, with the portable easyCyte occupying a volume similar to that of a desktop printer. Piezoacoustic focusing, or inertial focusing via Dean flow in curvilinear channels, can be coupled to microfluidics to potentiate flow-on-a-chip. The NanoCellect WOLF is a benchtop cell sorter that isolates and collects cells under low pressure using microfluidic cartridges that are sterile and disposable. Microfluidics presents an opportunity for improved accuracy and safer fluidics in cell sorting and collection, if not in speed and efficiency. All in all, users now have a wide array of choices when it comes to fluidics in cytometers and cell sorters.