Learn About Flow Cytometry
Principle of flow cytometry
A beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (forward scatter or FSC) and several perpendicular to it (side scatter or SSC) and one or more fluorescent detectors. Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a higher wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors.By analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules, or the membrane roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.
Flow cytometers
Modern flow cytometers are able to analyze several thousand particles every second, in "real time", and some instruments, called fluorescence activated cell sorters, can actively separate and isolate particles having specified properties. 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 set parameters. To analyze solid tissues, a single-cell suspension must first be prepared.A flow cytometer has five main components:
- Flow cell - liquid stream carries and aligns the cells so that they pass through the light beam in single file for sensing.
- Optical system - commonly used are lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers (argon, 488 nm; red-HeNe, 633nm; green-HeNe; HeCd, UV); and diode lasers (blue, green, red, violet) resulting in light signals.
- Detector and analogue-to-digital conversion (ADC) system - translates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer.
- Amplification system - linear or logarithmic.
- Computer - for analysis of the signals.
- Robotics for manipulation of multiple tubes and multi-well plates
Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for commercial instruments, as well as the reagents used in analysis, such as fluorescently-labeled antibodies and analysis software.
Modern instruments usually have multiple lasers and fluorescence detectors (the current record for a commercial instrument is 4 lasers and 18 fluorescence detectors). Increasing the number of lasers and detectors allows for multiple antibody labelling, and can improve precision. 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.
Reagent choice and experimental design
The specific setup of lasers and detectors of a particular flow cytometers determines which wavelengths are available to excite which fluorophores, and which wavelengths can be detected. The photomultiplier (PMT) voltage, type of signal amplification, available filters and other optical parameters also affect fluorophore choice. Instrument manufacturers typically provide excellent support for fluorophore selection processes.Fluorophores should be chosen with optimum signal compared to background. Sources of background include autofluorescence (in the absence of labeled antibody), electronic noise, and spillover fluorescence from other fluorophores. Autofluorescence is highest at high wavelengths (around 600 nm), so for highly autofluorescenct cells, it may be helpful to choose fluorophores with emission spectra at lower wavelengths. One way to evaluate signal-to-noise for any fluorophore is to calculate the signal width D/W, where D is the separation of positive and negative populations, and W is equal to the two standard deviations from the center of the negative population. Optical properties of fluorophores affecting the strength of the signal include:
- Extinction coefficient
- Quantum yield
- Stokes shift
- Spectral overlap (important for multicolor experiments)
Fluorophores have undergone many technological enhancements since their first introduction to flow cytometry. Traditional dyes (such as FITC, rhodamines, or phycobiliproteins) suffered from either low sensitivity or large molecular weight. Modern dyes such as Alexa or cyanine dyes offer a wide choice of emission spectra as well as low molecular weight (for minimal perturbation to experimental biomolecules).
Choosing the optimal fluorophore, though, also depends on the cell type, the antibody and antigen being detected. For some fluorophores, a single fluorophore molecule is conjugated to each antibody. But for others, multiple fluorophore molecules are conjugated to each antibody. For detecting rare antigens, it may be beneficial to choose fluorophores that are conjugated 1:1 to the antibody, to ensure separation between positive and negative cell populations. As some antibody-fluorophore combinations show higher nonspecific binding than others, choosing the best reagent may require testing several reagents.
Flow cytometry data output
The data generated by flow cytometers can be plotted in a single dimension, in a histogram, in two- dimensional dot plots, or even in three dimensions. The regions on these plots can be sequentially separated based on fluorescence intensity by creating a series of subset extractions, termed "gates". Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology. Often, data accumulated using the flow cytometer can be re-analysed elsewhere, freeing up the machine for other people to use.Flow cytometry data were plotted on logarithmic scales. However, because logarithmic scales are asymptotic to zero, zero or negative values could not be accurately displayed. Since these values are real (usually result of subtracting background or of compensation, which will be discussed below), bi-exponential (“logicle”) scales are now used to plot data. On a bi-exponential scale, the region close to zero approximates linearity, allowing the user to visualize zero or negative data points, enabling correct compensation.
Advanced Flow Cytometry Topics
ControlsTo obtain the maximum, highest-quality information from a flow cytometry experiment, users should include samples with known expected signals to use as positive, negative and instrument controls.
- Bead/microsphere control: Instead of cells (which can vary in size and shape), use a sample of extremely uniform standard reference particles, such as beads, to adjust the PMT voltage settings on the instrument. The instrument is set up properly when the scatter from the beads falls in the same place for every color channel.
- Fluorescence Minus One (FMO) control: Stain cells with all reagents except the one of interest. This is the best way to determine the boundary between positive and negative cells, and is especially important for dim populations.
- Experimental controls: cells that do not harbor the perturbation presumably being measured by flow cytometry. For example, in an experiment measuring receptor internalization in response to drug, include cells that have not been exposed to drug. This control will also enable the user to correct for autofluorescence, which should be identical for both treated and untreated cells.
- Compensation controls: cells stained with one color fluorophore conjugated to its specific antibody and the remaining fluorophores conjugated to non-specific isotype controls. More about compensation below.
Because different fluorescent dyes' emission spectra overlap [1], signals at the detectors have to be compensated electronically as well as computationally when acquiring data from a multicolor flow cytometry experiment.
- Electronic (“hardware”) compensation: The flow cytometer instrument contains a detector that senses spillover from one fluorescence wavelength to another. Before digitization of the fluorescence signal, the spill detector causes a fraction of the primary analog signal to be subtracted by differential signal amplification.
- Digital (“software”) compensation: Depending on the amount of spectral overlap between the different dyes, the digital signal from individual dyes can be multipled by a compensation coefficient, which is proportional to the inverse of the spectral overlap.
Compensation technique
Flow cytometry software allows the user to set the compensation coefficients by visualizing spillover from cells labeled with each dye individually (For example, the user determines how much yellow signal is spilling over from a green-labeled cell population.) Correct compensation requires that:
- PMT voltage is set correctly so that cells labeled with only isotype controls fall outside the “positive” region of the dot plot
- Analysis gate is set to compare cells with the same autofluorescence
- The median fluorescences of the labeled and unlabeled cell populations are aligned.
Staining methodology
Protocols for treating cells with fluorophore-conjugated antibodies must take into account several factors:
- Optimal antibody concentration -- first test a range of concentrations to find the one that provides maximum specific staining with minimal nonspecific staining, without reaching the point of saturation (binding to all specific AND nonspecific sites).
- Time
- Temperature
- Compatibility with protocol for other antibodies used in a multi-color experiment
- Order of addition (when using several antibodies) to accommodate epitope accessibility
Strategies for designing multicolor experiments
- Try to choose the brightest fluorophores for the least expressed proteins, and the dimmest fluorophores for the most expressed proteins.
- If there is no expression data for the antigens, assign the brightest fluorophore to the most important marker.
- Choose fluorophores excited by different lasers before choosing fluorophores excited by the same laser – less compensation will be required.
We obtained much helpful information from:
James W. Tung, Kartoosh Heydari, Rabin Tirouvanziam, Bita Sahaf, David R. Parks, Leonard A. Herzenberg, and Leonore A. Herzenberg. Modern Flow Cytometry: A Practical Approach. Clin Lab Med. Vol 27, p. 453 (2007)
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