Flowcytometry Basics

The measurement or quantification of cells suspended in a fluid phase is performed using flow cytometry. Fluorochrome-coupled antibodies specific for a certain cellular marker are used to label the cells. The fluidics chamber enables that cell travel via a laser beam in a single file, exciting the fluorochrome. A detector (photomultiplier) detects the produced light, which is then electronically translated and sent to a computer for analysis. Flow cytometry enables for the measurement of protein expression on a per-cell level, making it measurable with results presented as a count or number of events because of its single-cell nature.

The necessity to evaluate protein expression and phenotype living cells prompted the development of flow cytometry. Using microscopy to visualize and identify cells was insufficient; a method to assess phenotypic changes in live cell mixtures based on biological markers was required. Cell sorting was born when mixed populations of living cells required to be segregated or sorted based on phenotype in addition to cell identification. Instrumentation, monoclonal antibodies, and fluorochrome are the three main components of flow cytometry.

Components of Flow Cytometry

Flow cytometer instrument: A fluidics system intended to transport cells through the instrument, one or more lasers to excite fluorochromes, an optics system to detect light generated by the excited fluorochromes, and software to interpret the gathered data into a user-friendly readout make up fluidics-based flow cytometers.

Antibodies for flow cytometry: Any flow cytometry experiment relies heavily on the availability of potent, specific, and sensitive antibodies. To detect proteins in their normal, fully folded structure on a live or fixed cell, flow cytometry antibodies are necessary. Any antibody used in flow cytometry investigations must be thoroughly verified and described utilizing a variety of approaches. ELISA, Western blot, Immunocytochemistry (ICC), Immunohistochemistry (IHC), biological cell models, transfected cell lines, knockouts, and orthogonal approaches are some of the methods used to validate antibodies for flow cytometry.

Fluorescent dyes: Without a huge number of fluorescent dyes conjugated to antibodies, flow cytometry would not be possible.

Evolution of Flowcytometry

Cellular Impedance and the Coulter Principle

The notion of cellular impedance, a technique for counting cells based on the Coulter principle, was first patented by Wallace H. Coulter in 1953. Mack Fulwyler then utilized the Coulter principle to create a cell sorting apparatus. Different-sized cells in droplets flowed through an electrostatic field, which was utilized to sort cells into collecting bins based on their size differences. Although cell sorting has progressed significantly, the same approach of moving cell-containing droplets via an electric field is still used today.

Fluorescence-Based Flow Cytometers

Wolfgang Göhde of the University of Münster invented the first fluorescence-based flow cytometer in 1968, which sorted cells based on fluorescence rather than cell size. The creation of the flow cell, which allowed cells to be directed into a focus point and studied individually, was one of Göhde’s most important contributions to the discipline of cytometry. The use of fluorescent molecule emission rather than absorption, which was more popular at the time, was another significant innovation.

Monoclonal and Polyclonal Antibodies

The development of monoclonal antibodies and fluorescent molecules was prompted by the emergence of fluorescence-based flow cytometers. Polyclonal antibodies are antibodies that bind to several epitopes of the same target antigen generated by different B cells.

Polyclonal antibodies are often generated in larger animals and purified from their serum rather than immortalized hybridomas that produce monoclonal antibodies. Because polyclonal antibodies are often made from entire proteins, cross-reactivity between various isoforms of the same protein can occur, making a polyclonal antibody less selective than a monoclonal antibody.

Monoclonal antibodies, unlike polyclonal antibodies, may be mass-produced in huge quantities with high lot-to-lot consistency and excellent epitope specificity. Monoclonal antibody genes are extracted from B cells, amplified, and cloned into the appropriate vector system, allowing for infinite antibody production by bacterial or mammalian cell lines.

Fluorescent Molecules

In 1942, the “florescent antibody hour” began. After struggling with anthracence isothiocynanate, Coons and colleagues switched to fluorescein iso-cynanate, often known as FITC, to identify antiPneumococcus antiserum. They discovered that by tagging tissues infected with strain 3 Pneumococcus, they could detect fluorescent staining but not strain 2, demonstrating one of the first uses of an antigen-specific fluorescently tagged antibody.

Fluorochromes are luminous chemical substances that produce light at a certain wavelength when excited. Fluorochromes are stimulated with laser light of a certain wavelength in classical flow cytometry. The photomultiplier tube of the cytometer detects light emitted by the excited fluorophore at a certain emission wavelength.

Fluorescence Activated Cell Sorting (FACS)

Leonard Herzenberg rapidly there was a need to expand on the concept of sorting live cells based on fluorescence. Monoclonal antibodies specific to markers on the cells of interest paired with fluorescent molecules enabled fluorescent sorting of cells depending on the phenotypic of surface markers, a technique pioneered by the Herzenberg lab. Dr. Herzenberg invented the term “FACS,” or Fluorescence Activated Cell Sorting, to describe the process of sorting cells that uses fluorescence rather than size.

The word FACS is frequently misunderstood to mean collecting cells on a flow cytometer and seeing them on multicolor flow plots, whereas it actually refers to the physical separation of cells based on electrical charge and antibody fluorescence. The initial FACS apparatus had a laser and two light detectors, one for measuring cell size and the other for measuring fluorescence. Becton Dickinson (BD) Biosciences created the first commercial FACS apparatus in 1974, followed by the Partec/Phywe (ICP 22, 1975) and Epics from Coulter (1977).

Modern Flow Cytometers

There are three types of flow cytometers available on the market today:

  1. Fluidic-based flow cytometers.
  2. Acoustic-based flow cytometers.
  3. Mass cytometers or CyTOF.

Fluidic-Based Flow Cytometers

Flow cytometers based on fluidics are widely used in laboratories all over the world. More lasers and complicated optics have been added to flow cytometers as they have advanced, allowing for more parameters to be evaluated simultaneously. Initially, flow cytometers were equipped with one or two lasers: a blue laser (488 nm) and a red laser (633 nm). Fluorochromes such as Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), and Peridinin-Chlorophyll Protein Complex (PerCP) can be excited by the blue laser, while Allophycocyanin can be excited by the red laser (APC). In the early 1990s, photomultiplier detection of these four fluorophores served as the foundation for flow cytometry investigations.

In the early 2000s, the addition of the violet laser (405 nm) and the UV laser (350 nm) opened up the formerly underused portion of the light spectrum, paving the door for the development of novel dyes excitable at these wavelengths. The recent addition of a fifth laser, the yellow-green (561 nm), enables for the excitation of PE by a different laser than that used to excite FITC, considerably lowering the need for spectral overlap adjustment between these two fluorochromes. Researchers benefit from the low cost and availability of fluidic-based flow cytometers.

Components of Fluidic-Based Flow Cytometers

Fluidics

A flow cytometer’s fluidics system is an essential component. It is in charge of conveying fluorescently labeled cells to the probing point so that they can be activated by the lasers. This is performed by the use of hydrodynamic focusing, which is achieved by generating laminar flow, which occurs when two streams of fluid with differing flow rates run side by side in the same direction.

Fig: Hydrodynamic focusing Two streams of fluid—cells in sample fluid and cells in sheath fluid—move side by side into the flow cell, creating a laminar flow that focuses the cells in the flow cell nozzle, forcing them into single file.

Optics

Following hydrodynamic focusing, the cells enter the optics system in a single file. The optics system is made up of two parts: the Excitation Optics system, which uses laser beams to scatter light and excite fluorochrome-labeled antibodies on cells, and the Collection Optics system, which uses filters and mirrors to direct the scattered and emitted fluorescent light to a series of Photomultiplier Tubes (PMTs). PMTs, also known as detectors or channels, digitally convert light into data points that are graphically shown in flow graphs on a computer.

The Forward Scatter Channel (FSC) collects forward scattered light and uses it to calculate cell size while also accounting for nuclear to cytoplasmic ratio, membrane architecture, and other cellular properties. Side Scatter (SSC) is light that is scattered perpendicular to the laser beam and offers information on cellular granularity and complexity. In addition to the information gathered by the FSC and SSC channels, the optics system is in charge of filtering and directing fluorescent light emitted by fluorochromes to the PMTs that are particular to certain fluorochromes.

Electronics

The electronics system turns the emission spectra into digital signals that the computer may analyze using software such as BD FACSDiVaTM or other acquisition tools available on the market. Analog flow cytometers, such as the BD FACSCaliburTM, predate digital flow cytometers, such as the BD LSRFortessaTM. Analog to Digital converters, (ADC), digitize voltages into one of 16,384 potential levels 10 million times/s using the BD LSRFortessaTM.

During the amplification and digitization process, voltage pulses are binned and allocated a height, width, and area. The resolution of ADCs varies depending on the instrument, although higher-bit ADCs have better resolution. The ADC resolution is a measure of the number of bins to which the data may be assigned. Signals are then sent to the computer through Ethernet wires in order to show the data graphically. For FSC/SSC and fluorescent spectra, the electronics measure the area, height, and breadth parameters. Flow data plots are the outcome of the Flow Cytometer’s electronics converting these fluorescent signals into usable data.

Three main plots routinely used include the following:

I) Dot plots: Two parameter plots that illustrate

  • cell size/granularity based on FSC and SSC or
  • protein expression on cells using fluorochrome-conjugated antibodies.

Individual cells or dots are used to represent the data, which can be displayed using monochromatic dot plots (e.g., black) or pseudocolor dot plots. Dot plots are similar to heat maps in that they indicate the density of each cell population.

II) Histograms: One parameter plot that depicts cell number on the y-axis vs fluorescence or protein expression on the x-axis.

III) Density plots: Two parameter graphs demonstrating the accumulation and relative density of cells expressing the proteins of interest. The data is shown as contours (rather than individual cells as in dot plots), with high/dense contouring indicating a large number of cells expressing the proteins of concern.

Acoustic-Based Flow Cytometers

Life Technologies’ Attune NxT is an acoustic-based flow cytometer. The equipment transports cells to the center of the sample stream using acoustic focusing and ultrasonic radiation pressure before injecting them into the sheath stream. This technique allows for a small core stream and consistent laser illumination. As a result, the cytometer is less susceptible to blockages and can analyze up to 35,000–50,000 events/s, whereas fluidic-based cytometers typically run at around 3000 events/s.

The Attune can manage up to 14 parameters, and autosamplers can run up to 384 samples in a plate-based fashion. As the identification of uncommon occurrences such as tumor cells in whole blood becomes more relevant in medical diagnosis and therapy, large analysis flow rates employing acoustic flow cytometry can help. Because these equipments are more expensive, they may be out of reach for certain researchers.

Mass Cytometers (CyTOF)

The HeliosTM CyTOF (Cytometry by Time of Flight) from Fluidigm combines the best of flow cytometry and mass spectrometry. CyTOF, which was introduced in 2014, uses monoclonal antibodies tagged with heavy metal tags rather than fluorochromes. Following labeling, the cells are sprayed as single droplets in an argon plasma (5000 °C), which is subsequently evaporated, and the electrons from the metals are removed to create ions. The ions are propelled into an electric field, and a deflector separates them as they fly toward the collecting screen at varying rates determined by their mass.

The ions are quantified by the detector, and the data is evaluated and presented on the computer. Because the samples are cremated, sorting is not feasible with this mass cytometer; nonetheless, it can assess 50–100 markers at a time. Whereas spectrum overlap must be corrected in fluidic/acoustic flow cytometry, CyTOF needs less adjustment due to the use of heavy metal tags rather than fluorochromes. Flow rates are substantially slower (500 events/s) than those of fluidic and acoustic flow cytometers (3000 and 35,000, respectively).

Furthermore, the HeliosTM CyTOF cytometers are rather costly. When this is combined with the additional expense of conjugating antibodies to heavy metal tags, most researchers will find this approach too expensive. This method is especially beneficial in clinical contexts when a high number of variables may be examined with a small sample size.

Fig. The physics of Fluidigm’s HeliosTM CyTOF. A diagram demonstrating how the CyTOF works. Cells tagged with antibodies attached to heavy metals are evaporated and ionized, after which the ions are accelerated in an electrostatic field, separated by a deflector, and counted based on their mass.

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