Flow cytometry

A flow cytometer can tell us a cell's size, granularity/complexity and its fluorescence intensity, relative to other cells in the sample and to the controls.

The fundamental strength of the technology is its ability to captures many parameters of each individual cell, combined with its ability to process cells very quickly. This is unlike bulk analysis.

For example, flow cytometry can rapidly count the number of cells secreting a certain protein; a bulk analysis method can just roughly measure the total amount of secreted protein for a population.

History

The first practical applications of flow cytometry were in the 1940s, to count blood cells in liquid suspension and bacteria and other small particles in aerosols.

In the early 1960s, light absorption measurements were used fro quantitative flow cytometric analyses of cellular nucleic acid and protein. The first fluorescence flow cytometers were built in the late 1960s.

Fundamental Steps

Flow cytometers all have three steps: uptake, interrogation and effluence. Essential to these are sample preparation and analysis.

Uptake

A probe uptakes a liquid sample and channels it into a flow cell that focuses the cells into a single file stream.

This stream is surrounded by sheath fluid, so called because it sheaths the sample in a fluid known to have nonturbulent, laminar flow (usually PBS).

Interrogation

A laser shines through one cell at a time.

The flow passes through one or more light sources. Lasers are favored because they have consistent wavelength and direction. A detector receives the light that passes through each cell and converts it into an electronic signal to send along to the computer.

The most common type of detector in flow cytometry is a photomultiplier tube (PMT).

The simplest flow cytometer has only one light source, with two detectors.

One detector measures forward scatter (FSC) (cell size) and the other measures side scatter (SSC) (cell complexity). The cell will diffract some light, bending it away from the incident angle; a detector at a ~5° angle to the incident light will detect this, which is proportional in magnitude to the cell size.

Intracellular components will also scatter some light; a detector at a 90° angle to the incident light will detect this, which is proportional in magnitude to the cell's complexity.

To detect fluorescence, flow cytometers have additional sets of light sources and detectors.

The incident light has a certain wavelength. This is the excitation wavelength because when it strikes each cell it excites labels that in turn fluoresce at a different emission wavelength. The flow cytometer relies on the Stoke's shift, the phenomenon where a substance absorbs light at one wavelength and emit it at another.

This is how the flow cytometer discerns between light from the source and light from a positively labeled cell. The use of fluorescent-labeled antibodies allows the flow cytometer to discern labeled subpopulations.

The fluorochrome must have the same excitation wavelength as the incident light, and an emission wavelength detectable by the detector.

Some labels share excitation wavelengths but have different emission wavelengths. To discern these, one laser can have multiple detectors. Each detector has a filter (swappable in newer machines) so that it will only detect a specific wavelength (color).

The rise of flow cytometry continues hand-in-hand with the availability of antibodies for increasingly specific subpopulations, and the development of new fluorescent molecules (fluorochromes).

Effluence

Subsequently, the cells are either put into a waste container or sorted.

Cell Sorting

Fluorescent-activated cell sorting (FACS) refers to this same process but with additional components that physically separate cell subsets based on one or more parameters.

Sample Preparation

Flow cytometers uptake single cell suspensions.

They excel most when the least preparation is required to achieve a single cell suspension: namely, flow cytometers are best at analyzing blood samples. It is possible to dissociate tissues and even whole organisms into suspensions of intact cells, but removal of the adhesion molecules is a huge loss; these are likely as important as anything else for understanding the cells.

Also, the observation (a few hundred milliseconds) is very short.

Analysis

The flow cytometer outputs raw signals from the various detectors. These signals are presented using computer software and in turn analyzed by the researcher.

This is mostly done with plots and histograms.

Determining Cells

Any particle that is picked up by a detector is called an event.

Event is a vague term which underlines the need to analyze whether an event is debris, contamination or part of the population of interest. The trigger signal is the threshold at which an event vs non-event is determined.

Crystals and other debris will send pulses of light to the detectors as the sheath and suspension fluids pass through the interrogation zone. To discern between cells and non-cells, a threshold is set: any pulse greater than a certain value will be collected, while any pulse lower will not be recorded. Also, doublet discrimination is crucial.

Main Population

If enough events have similar characteristics then they will form distinct populations that can be detected on a dot-plot or histogram.

Consider a sample with one cell type, and you want to measure its response to a chemical that induces apoptosis. You have an untreated negative control, and then a treated sample. You label both with an antibody specific for cell surface antigens present during apoptosis. Running these samples through the flow cytometer, you see lots of various events but there seems to be a high number of cells with certain forward scatter (FSC) and side scatter (FSC) characteristics. This is your population of interest.

You have the software then return to you the fluorescence information on this population of interest. The negative control has some autofluorescence but the treated sample fluoresces several magnitudes stronger. This is an ideal, simple one-color flow cytometry experiment.

Flow cytometry applications

Flow cytometry systems

Particle analysis	Size distribution Refractility Anemia Diagnosis Leukemia Diagnosis Detection of Infection Concentration
Sorting	Cell enrichment Cell concentration Verification of analysis Antibody production Toxicity studies
Cell populations	Flow cytometry can detect cell populations, whether endogenous, infectious, oncogenic or otherwise. Hoescht Side PopulationIn an otherwise homogeneous population, Hoescht staining can identify cells with elevated metabolisms. In this application, it is usually used to determine percentages of oncogenic or stem cells (particularly iPSCs). Cell classification Clone enumeration Hoescht Staining (Stem Cells) Proliferation rate Variant DNA quantitation Leukemia typing Diagnosis Infection
Cell Biology, Cycle and Activity	Intracellular stains and antibodies enable cell biology analysis. Cell SizeForward scatter (no need for staining) Cell shapePulse shape analysis Protein content Enzyme activityChromogenic or fluorogenic substrates (the enzyme's activity on the substrate changes the substrate's spectral properties) Membrane-Bound Ca²⁺Fluorescent chlortetracycline. Cytoplasmic [Ca²⁺]Look at the fluorescence ratio, usually via Indo-1; or the fluorescence, usually via Fluo-3 Intracellular pHLook at the fluorescence ratio, usually via ADB, BCECF, SNAFL or SNARF. Cell cycleModFit is the software usually used to study flow cytometry cell cycle analysis. Cell biochemistry Mutagenesis Chemotaxis Genetic disorders Cell toxicity Cell function Cell activity EndocytosisLabeled beads or bacteria Chromosome analysis Cell structure Organelles Cytoplasm-Mitochondria membrane potentialFluorescence of cyanines, oxonols, rhodamine 123
DNA, RNA and Genomics	DNA QuantityDAPI and especially DNA Propidium Iodide (PI) are two popular DNA stains. The former requires a UV laser while the latter may be run on almost any flow cytometer. Since DNA content corresponds to cell cycle phase, these stains are usually used for such studies. It can be roughly deduced without a stain based on UV absorption at 260 nm in unstained cells. DNA base compositionA-T and G-C preference dyes such as Hoechst 33342 and chromomycin A₃, cyanine/styryl dyes DNA synthesisFluorescence of anti-BrUdR antibodies or SBIP DNA degradation (apoptosis)Labeled nucleotides ssRNA and dsRNA ContentAcridine orange, pyronin Y, thiazole orange Chromosome karyotypingUsing DNA base stains, it is possible to identify and sort most of the human chromosomes. This technique has been used as an early step in preparing the DNA libraries need to map the human genome.
Membrane (Lipid Bilayer)	Surface SugarsLabeled lectins bind at lectin binding sites. Membrane potential Viability (membrane integrity)Propidium iodide (PI), fluorescein diacetate (FDA), trypan blue Membrane fluidityFluorescence polarization (DPH)
Immunology	Epitope density Epitope type Surface receptor proximity Lymphocyte subsets Cell surface structure Study of immune system Predisposition to diseases
Virology	TiterTo determine the proper titer of a virus, commonly a GFP construct is included. After transfecting cells at various titers, GFP expression is then assayed using a flow cytometer to find the optimal viral concentration where most cells are transfected.

Fluidics	Hydrodynamic focusing Microliter flows Fluid jets Droplet dynamics Sheath fluidThe fluid that surrounds the stream. It is nonturbulent and has laminar flow. Usually it is just phosphate buffer solution (PBS) carefully prepared with filtered, sterile contents; for sorters, it must be purchased and sealed to ensure sterility. Measurement regionThe fluidics system flows through the machine, and the measurement region is the segment where particles are interrogated by lasers. Also known as the measurement, analysis, interrogation or observation point, station or zone; EffluenceEffluence refers to the mixture of sheath fluid and sample intake after interrogation. Waste TankAnalytical (non-sorting) machines pour the effluence into a waste tank that usually contains bleach to the sterilize the effluence for sink disposal. The tank must be emptied regularly lest it overflow and leak (which can corrode the machine). Sorting
Pneumatics	Air pumpIt is easier to limit extra pressure than to have a consistently perfect pump. Essentially an aquarium pump, it outputs high pressure that is then pared to a precise value and controlled to a smooth consistency. Air pressure control
Optics	Lasers Arc lamps Light scatter Fluorescence Ultra sensitive sensorsVoltage may be adjusted, thereby controlling sensitivity and thus the apparent intensity of the signal. Polarization Lenses Sharp cutoff filters Long pass filters Bandwidth filters
Electronics	Low noise pulse preamps High speed log amplifiers 100MHz A/D converters Real-time control logic Quiet Kilovolt supplies Stroboscopic illumination
Computers and Software	Real-time data acquisition Megabyte data streams Multichannel analyzers Multivariate analysis Pattern recognition Graphical User Interface (GUI) Data Analysis
Electrostatics (Sorting)	Charge control Droplet deflection
Ultrasonics	Rayleigh distortion Jet-droplet formation
Reagents and Cells	Fluorescent stains Monoclonal antibodies Reaction kinetics Epitope density Steric hindrance Matrix interferences Beads (polymer microspheres) Cell physiology Osmolarity Physiological pH Shear stress Temperature control

Studies

Darzynkiewicz, et al. 2005. Essential cytometry methods. Oxford, UK: Academic Press.

Givan, A L. 2001. Flow cytometry: first priciples. New York, NY: Wiley-Liss.

McCarthly, D A and Macey, M G. 2001. Cytometric analysis of cell phenotype and function. Cambridge, UK: Cambridge University Press.

Rose, N R. Manual of clinical laboratory immunology (American Society for Microbiology, 1986). Chapter 32:

Preparation, Staining and Analysis by Flow Cytometry of Peripheral Blood Leukocytes.

Haynes, John. Principles of Flow Cyteomtry. Becton Dickinson Research Center. (This has the excellent index of flow cytometry technologies from which I elaborated.)