Flow cytometry


Flow cytometry is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer.
Flow cytometry is routinely used in basic research, clinical practice, and clinical trials. Uses for flow cytometry include:
A flow cytometry analyzer is an instrument that provides quantifiable data from a sample. Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties.

History

The first impedance-based flow cytometry device, using the Coulter principle, was disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. Mack Fulwyler was the inventor of the forerunner to today's flow cytometers – particularly the cell sorter. Fulwyler developed this in 1965 with his publication in Science. The first fluorescence-based flow cytometry device was developed in 1968 by Wolfgang Göhde from the University of Münster, filed for patent on 18 December 1968 and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still widely favored by other scientists over fluorescence methods. Soon after, flow cytometry instruments were developed, including the Cytofluorograph from Bio/Physics Systems Inc., the PAS 8000 from Partec, the first FACS instrument from Becton Dickinson, the ICP 22 from Partec/Phywe and the Epics from Coulter. The first label-free high-frequency impedance flow cytometer based on a patented microfluidic "lab-on-chip", Ampha Z30, was introduced by Amphasys.

Name of the technology

The original name of the fluorescence-based flow cytometry technology was "pulse cytophotometry", based on the first patent application on fluorescence-based flow cytometry. At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola in 1976 – eight years after the introduction of the first fluorescence-based flow cytometer – it was agreed to commonly use the name "flow cytometry", a term that quickly became popular.

Flow cytometers

Modern flow cytometers are able to analyze many thousands of particles per second, in "real time" and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers high-throughput, automated quantification of specified optical parameters on a cell-by-cell basis. To analyze solid tissues, a single-cell suspension must first be prepared.
A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream, which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system commonly uses measurement of impedance and optical systems – lamps ; high-power water-cooled lasers ; low-power air-cooled lasers, red-HeNe, green-HeNe, HeCd ); diode lasers resulting in light signals. The detector and analog-to-digital conversion system converts analog measurements of forward-scattered light and side-scattered light as well as dye-specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be linear or logarithmic.
The process of collecting data from samples using the flow cytometer is termed "acquisition". Acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer. The software is capable of adjusting parameters for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in acquisition such as fluorescently labeled antibodies have been developed.
Modern instruments usually have multiple lasers and fluorescence detectors. The current record for a commercial instrument is ten lasers and 30 fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their phenotypic markers. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells.

Hardware

Fluidics system of a flow cytometer

Cells must pass uniformly through the center of focused laser beams to accurately measure optical properties of cells in any flow cytometer. The purpose of the fluidic system is to move the cells one by one through the lasers beam and throughout the instrument. Fluidics in a flow cytometer with cell sorting capabilities also use the stream to carry sorted cells into collection tubes or wells.

Hydrodynamic focusing

For precise positioning of cells in a liquid jet, hydrodynamic focusing is used in most cytometers. The cells in suspension enter into the instrument enclosed by an outer sheath fluid. The sample core is maintained in the center of the sheath fluid. The sample input rate or how fast the cells flow through to the laser interrogation can be controlled by the pressure of the sheath fluid on the sample core. Under optimal conditions, the central fluid stream and sheath fluid do not mix.

Acoustic-assisted hydrodynamic focusing

Acoustic focusing technology is used in some flow cytometers to support hydrodynamic focusing. Acoustic waves pre-focus the sample before introduction to sheath fluid. The pre-focused sample is then injected into the hydrodynamic core and flowed through the instrument. This may help with increasing data accuracy under high sample input rates.

Optics and electronics

Optical filters

Light emitted from fluorophores are in a spectrum of wavelengths, so combining multiple fluorophores may cause overlap. To add specificity, optical filters and dichroic mirrors are used to filter and move light to the detectors such as photomultiplier tubes or avalanche photodiodes. Optical filters are designed as band pass, long pass, or short pass filters. Most flow cytometers uses dichroic mirrors and band pass filters to select specific bands of the optical spectrum.

Prisms, gratings, and spectral flow cytometry

Spectral flow cytometry uses prisms or diffraction gratings to disperse the emitted light of a marker across a detector array. This allows for the full spectra from each particle to be measured. The measured spectra from single cells are subsequently unmixed by using reference spectra of all used dyes and the autofluorescence spectrum. This may allow for a wider panel design and the application of new biological markers.

Imaging flow cytometry

Imaging flow cytometry captures multichannel images of cells. Detectors used in imaging platforms can be equipped with charge-coupled device or complementary metal–oxide–semiconductor to capture images of individual cells.

Data analysis

Compensation

Each fluorochrome has a broad fluorescence spectrum. When more than one fluorochrome is used, an overlap between fluorochromes can occur. This situation is called spectrum overlap, and must be corrected. For example, the emission spectrum for FITC and PE is one in which the light emitted by the fluorescein overlaps the same wavelength as it passes through the filter used for PE. This spectral overlap is corrected by removing a portion of the FITC signal from the PE signals or vice versa. This process is called color compensation, which calculates a fluorochrome as a percentage to measure itself.
Compensation is the mathematical process by which spectral overlap of multiparameter flow cytometric data is corrected. Since fluorochromes can have wide-ranging spectrum, they can overlap, causing the undesirable result of confusion during the analysis of data. This overlap, known as spillover and quantified in the spillover coefficient, is usually caused by detectors for a certain fluorochrome measuring a significant peak in wavelength from a different fluorochrome. Linear algebra is most often used to make this correction.
In general, when graphs of one or more parameters are displayed, it is to show that the other parameters do not contribute to the distribution shown. Especially when using the parameters which are more than double, this problem is more severe. Currently, no tools have been discovered to efficiently display multidimensional parameters. Compensation is very important to see the distinction between cells.
File:Picoplancton cytometrie.jpg|thumb|upright=1.25|right|Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations