Flow cytometry

Traditional Flow Cytometers 

Traditional flow cytometers consist of three systems: fluidics, optics and electronics. The fluidics system consists of sheath fluid (usually a buffered saline solution) that is pressurized to deliver and focus the sample to the laser intercept or interrogation point where the sample is analyzed. The optical system consists of excitation optics (lasers) and collection optics (photomultiplier tubes or PMTs and photodiodes) that generate the visible and fluorescent light signals used to analyze the sample. A series of dichroic filters steer the fluorescent light to specific detectors and bandpass filters determine the wavelengths of light that are read so that each individual fluorochrome can be detected and measured. More specifically, dichroic filters are filters that pass light through that is either shorter or longer in wavelength and reflect the remaining light at an angle. For example, a 450 Dichroic Long Pass filter (DLP) lets light that has a longer wavelength than 450 nm through the filter and bounces the shorter wavelengths of light off at an angle to be sent to another detector. Bandpass filters detect a small window of a specific wavelength of light. For example, a 450/50 bandpass filter passes fluorescent light that has a wavelength of 450 nm +/− 25 nm through the filter to be read by the detector. The electronic system converts the signals from the detectors into digital signals that can be read by a computer.

Multiple laser systems are common with instruments often having 20 parameters (FSC, SSC and 18 fluorescent detectors). There are new instrument platforms being introduced with five or more lasers and 30–50 parameters, but these are less common. The most common lasers used in traditional flow cytometers are 488 nm (blue), 405nm (violet), 532nm (green), 552nm (green), 561 nm (green-yellow), 640 nm (red) and 355 nm (ultraviolet). Additional laser wavelengths are available for specialized applications. In addition, there are instruments that have replaced PMTs with avalanche photodiodes (APD) for fluorescence detection with the aim of increasing sensitivity.

Acoustic Focusing Cytometers 

This cytometer uses ultrasonic waves to better focus cells for laser interrogation. This type of acoustic focusing allows for higher sample input and less sample clogging. This cytometer can utilize up to 4 lasers and 14 fluorescence channels.

Cell Sorters 

A specific type of traditional flow cytometer is the cell sorter which can purify and collect samples for further analysis. A cell sorter allows the user to select (gate) on a population of cells or particles which is positive (or negative) for the desired parameters and then direct those cells into a collection vessel. The cell sorter separates cells by oscillating the sample stream of liquid at a high frequency to generate drops. The drops are then given either a positive or negative charge and passed through metal deflection plates where they are directed to a specific collection vessel based on their charge. The collection vessels can be tubes, slides or plates (96-well or 384-well are common).

There are two types of cell sorters, quartz cuvette and “jet-in-air” that differ in where the laser interrogation point is located. The quartz cuvette cell sorters have fixed laser alignment and are easier to prepare for a sort. The “jet in air” cell sorters need to have the lasers aligned daily and are more difficult to set up but are more adaptable for small particle detection.

Imaging Cytometers 

Imaging flow cytometers (IFC) combine traditional flow cytometry with fluorescence microscopy. This allow for rapid analysis of a sample for morphology and multi-parameter fluorescence at both a single cell and population level (Barteneva, Fasler-Kan, & Vorobjev, 2012). IFC can track protein distributions within individual cells like a confocal or fluorescence microscope but also to process large numbers of cells like a flow cytometer. They are particularly useful in multiple applications such as cell signaling, co-localization studies, cell to cell interactions, DNA damage and repair and any application that needs to be able to coordinate cellular location with fluorescence expression on large populations of cells.

Mass Cytometers 

Mass cytometers combine time-of-flight mass spectrometry and flow cytometry. Cells are labeled with heavy metal ion-tagged antibodies (usually from the lanthanide series) instead of fluorescently-tagged antibodies and detected using time-of-flight mass spectrometry. Mass cytometers do not have FSC or SSC light detection which does not allow for the conventional method of detecting cell aggregates. However other methods such as cell barcoding can be employed for this purpose (Leipold, Newell, & Maecker, 2015). Also, mass cytometry does not have cellular autofluoresce signals and reagents do not have the emission spectral overlap associated with fluorescent labels so compensation is not needed. However, the sample is destroyed during analysis so cell sorting is not possible and the acquisition rate is much lower than a standard flow cytometer (1000 cells/second instead of 10,000 cells/second). Currently, there are commercially available reagents for 40 channels but this number will increase with the introduction of other metal ions such as platinum for conjugation to antibodies (Mei, Leipold, & Maecker, 2016).

Cytometers for Bead Array Analysis 

Multiplex bead arrays have become popular for analyzing large amounts of analytes in small sample volumes. Briefly, these assays utilize capture beads with a known amount of fluorescence in a specific channel and a reporter molecule detected by a separate laser to quantify the amount of captured analyte associated with the specific bead. It is essentially the equivalent of 100 ELISA assays.

Small flow cytometers with usually 2 lasers and 96-well loaders have been developed to analyze these assays. These instruments have small footprints and optical bench designs that are optimized to detect and discriminate beads with different amounts of fluorescence along two channels. Instruments have been developed that can detect 100–500 different bead combinations.

Spectral Analyzers 

One of the challenges of multi-parameter flow cytometry is compensation (or erasing spectral overlap) between flurochromes. A new type of flow cytometer, the spectral analyzer is specifically designed to address this problem. A spectral analyzer measures the entire fluorescent emission spectra for each fluorochrome in a multicolor sample to create a spectral fingerprint. Then during analysis, each spectra is unmixed to provide a pure signal for each fluorochrome (Sony, 2017). Spectral analysis is starting to replace traditional PMTs as a detection method for high-dimensional flow cytometry.

New Detector Technologies 

Photomultiplier tubes (PMTs) remain the standard detector technology for flow cytometry. Their high sensitivity and low backgrounds make them useful for fluorescence technology. However, solid state detectors are starting to appear in some cytometers. Avalanche photodiodes (APDs) are inexpensive, sensitive and highly linear, and are more spectrally responsive in the long red region. Silicon photodiodes (SiPDs) are also a promising option for solid state.