Flow Cytometry

Flow cytometry allows a sample of cells or particles in suspension to be separated through a narrow, rapidly flowing stream of liquid. As the sample pass through a laser it allows for detection of size, granularity, and fluorescent properties of individual cells/particles in the sample. In this way, a composition of heterogeneous biological samples may be de-convoluted, such as identification of T-cells in blood or photosynthetic microbes in seawater. By using fluorescently labeled antibodies targeting carefully selected surface proteins an even finer insight of the sample may be achieved, e.g. differentiating activated and non-activated T-cell. In the following text the sample will be referred to as "the particle" and can be any particle or cell.

When particle passes through an opening concurrent with an electrical current the change in impedance is relative to the size of the particle and this is known as the Coulter principle. This is particularly useful when the particles have low conductivity, as the case with cells. Using the Coulter principle to count and measure the size of particles was implemented in the first cell sorter invented by Mack Fulwyler in 1965. The technique was expanded by Leonard Herzenberg who was responsible for coining the term FACS, fluorescent-activated cell sorter. The acronym FACS is trademarked and owned by Becton Dickinson. While many scientists use this term frequently for all types of sorting and non-sorting applications, it is not a generic term for flow cytometry.

Technology

A fluorescently labeled sample with particles of interests is connected to the flow cytometer where the particles will be oriented by hydrodynamic focusing into a thin stream surrounded by fluid of higher speed (sheet fluid). A beam of laser light is directed onto the stream of fluid and a number of detectors are aimed at the same point. The different detectors are sensing light in line with the light beam, Forward Scatter (FSC), and perpendicular to it, Side Scatter (SSC), as well as one or more sets of fluorescent molecules. FSC correlates with the particle volume, and SSC depends on the inner complexity of the particle. Typically, particles in the range from 0.2 to 150 micrometers passing through the beam scatters the light at a detectable level based on SSC or FSC. The scattered light in combination with fluorescent light from chemicals found inside the particle or attached on the particle surface, is identified by the detectors. Fluctuations in brightness at each detector (one for each fluorescent emission peak) are analyzed making it possible to derive various types of information about the physical and chemical structure of each individual particle. Modern instruments usually have multiple lasers and fluorescence detectors allowing for a more complex experimental setup. The process of collecting data from samples using the flow cytometer is termed 'acquisition'. Acquisition is mediated by a computer connected to the flow cytometer and the software that handles the digital interface with the cytometer. The software is capable of adjusting parameters (i.e. PMT voltage for amplification of signals, and compensation for leakage between fluorescent channels) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to insure that parameters are set correctly. Typically a flow cytometer has five main components before the sample collection, Figure 1:

1. a flow cell where the liquid stream (sheath fluid) which carries the cells/particles is aligned so that the cells/particles pass in a single file through the light beam for sensing
2. a measuring system, commonly used today include diode lasers of different wavelengths (blue, green, red, violet) resulting in light signals
3. a detector, most often Photo Multiplier Tube (PMT), and Analogue-to-Digital Conversion (ADC) system which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer
4. an amplification system (linear or logarithmic)
5. a computer for analysis of the signals

Figure 1. General principle for flow cytometry. A heterogeneous cell sample (A) is labeled by fluorescent antibodies (B) the sample particles are focused in a flow of fluid (C). The flow allows one particle at a time to reach a light source (D). Depending on the nature of the particle light will scatter or fluoresce at varying degree. This is monitored after signal amplification in a computer diagram where gates around particles of interest can be drawn (blue and red) (E). Certain flow cytometers are equipped with sorting capacity allowing gated particles with desired characteristics to be diverted into collection vessels (F).

Antibodies in Flow Cytometry

Antibodies are crucial tools for meaningful deconvolution of complex biological samples in flow cytometry as they allow pinpointing specific marker proteins on the cell surface. The more markers you use, the more information on each cell you get; the more antibodies you have the more information you can obtain. However, there are limitations both in number of different fluorochromes available and how many lasers and detectors you can fit into a FACS machine. Currently some of the most advanced flow cytometers carry seven lasers and 49 different detectors.

A recent technology called mass cytometry is circumventing some of the limitations with fluorochromes; instead antibodies are labeled with rare earth metals, which hence allow for simultaneous detection of 40 different antibodies at the time (Ornatsky et al., 2010).

Sorting by Flow Cytometry

Fluorescence-activated cell sorting (FACS) is a distinct type of flow cytometry. It allows single cell/particle sorting based on the detected light scattering or into tubes or microtitre plates, one cell at a time. The narrow stream of liquid (sheet fluid) carrying the cells allows for separation so that only one cell at the time reaches the detector. To allow isolation of specific cells in the flow, a vibrating mechanism is making the stream of cells break up into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through lasers and detectors determining scatter and fluorescence parameters. An electrical field is placed just at the point where the stream breaks into droplets. Based on the immediately-prior fluorescence intensity measurement a charge is applied, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into tubes or wells on a microtiter plate.

Specific Examples

Flow cytometry is routinely used in a number of research and diagnostic areas. In health care, flow cytometry is used to diagnose different diseases, including blood cancers, as well as a help in other clinical decision in the fields of transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting for sex pre-selection etc. In marine biology, the auto-fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterize abundance and community structure. In protein engineering, flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties.

Bacterial display and sorting can be used for epitope mapping (Rockberg et al., 2008). Antibodies are incubated with a library of bacteria, where each bacteria display a specific type of peptides on their surface for epitope mapping purposes. Two fluorescent signals are detected: antibody-peptide binding (blue fluorophores) and surface expression of normalization tag binding to pink control protein (red fluorophores). Some library members (Bacteria B and Bacteria C) do not express peptides recognized by the antibody and they will only allow for detection of the red fluorescence. To fish out the binder from the background of non-binding peptides a gate is drawn around the population of expressing and binding bacteria (marked with blue rectangle) and the flow sorter is set to collect these cells into a container (blue tube). Using the same principle the non-binding peptides can be sorted and collected into another container (red tube).

Figure 2. Identification and gating of cell populations for epitope mapping. Peptides (purple) are displayed on the surface of bacteria and incubated with labeled antibodies. Thereafter the bacteria are sorted based on their properties.

A typical result from an epitope mapping set up is shown in Figure 3 (Rockberg et al., 2008). A Human Protein Atlas (HPA) antibody targeting the cancer protein HER2 is incubated with a peptide library. Then bacteria expressing interesting peptides are isolated by drawing a gate (1) around them and running the flow sorter in sort-mode to collect them. The collected sample is then analyzed in the flow sorter; the enrichment of dots (bacteria) within the same region (2) indicates a successful sorting. The bacteria collected from gate 2 may then be sorted again and subjected to DNA sequencing in order to obtain the epitope peptide sequence expressed on their surface.

Figure 3. Epitope mapping of a HER2 antibody using bacterial display and cell sorting. An HPA antibody targeting the cancer protein HER2 is incubated with a peptide library and gating is applied to collect bacteria with specific properties (Rockberg et al., 2008).

References and Links

Ornatsky O et al., Highly multiparametric analysis by mass cytometry. J Immunol Methods. (2010)
PubMed: 20655312 DOI: 10.1016/j.jim.2010.07.002

Rockberg J et al., Epitope mapping of antibodies using bacterial surface display. Nat Methods. (2008)
PubMed: 19029907 DOI: 10.1038/nmeth.1272

Shapiro H, Practical Flow Cytometry, Wiley & Sons Inc 2003: http://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/flow-cytometry/practical-flow-cytometry/index.htm

Flow cytometry, a laser-based, biophysical technology used to count, measure size, and detect properties of particles in suspension: http://en.wikipedia.org/wiki/Flow_cytometry

Coulter counter, an apparatus for counting and sizing particles suspended in electrolytes: http://en.wikipedia.org/wiki/Coulter_counter

Mass cytometry, a mass spectrometry technique used for the determination of the properties of cells (cytometry): http://en.wikipedia.org/wiki/Mass_cytometry

Hydrodynamic focusing uses a liquid to create a tunnel from fluid in which the sample can be injected: http://en.wikipedia.org/wiki/Hydrodynamic_focusing

Antibodypedia - An open-access database of publicly available antibodies and their usefulness in various applications: http://www.antibodypedia.com