What is Flow Cytometry?
Flow cytometry is a popular and powerful analysis method in the biological sciences. The basic principle relies on laser-mediated activation of fluorescent proteins on the surface or inside a cell or particle. The huge difference compared to other laser-based analysis techniques is that the cells are not static (as in e.g. microscopy), but instead are travelling through a capillary system.
As the singularized cells pass by a laser beam, the size, granularity and fluorescence properties of each individual cell are detected by the scattered laser light as forward and sideward scatter (FSC and SSC). This signal is then translated into an electronic signal and visualized in the flow software. As most cytometers contain several different laser light sources, many different fluorophores can be recorded, referred to as multiparametric analysis. As the cells pass by the laser beams with relatively high speed, cytometers can analyze ~30.000 cells/second and still retain single cell resolution.
How is Flow Data analyzed?
The basis of flow data analysis is the visualization of fluorescence intensities on XY-plots. Each cell is represented by a dot placed as specific spot on the XY-plot. Correlations between fluorescent markers can easily be visualized and “gated” for. Gating is a tool with which one can separate a specific subpopulation from the rest of the data within one analyzed cell sample and visualize it on a separate plot. This allows the identification of rare events and rare subpopulations.
As the flow data also contains a time stamp for each analyzed cell, the visualization of fluorescent intensities over time is very easy and allows for the analysis of fluorescent changes over time. This capability makes flow cytometry very popular for kinetic analysis including calcium flux, membrane potential and ROS.
Flow Cytometry Applications
Any experiment working with fluorescent probes that does not require subcellular resolution can be analyzed with a classical flow cytometer. Below, we list a few very common assays of an ever-growing list of possible applications for flow cytometry.
Immunologists have relied on flow cytometry for the past decades to distinguish specific cell types in heterogenous cell populations (e.g. amount of CD4+ T cells within a blood sample). This is achieved by fluorescent-conjugated antibodies against surface molecules (and also intracellular proteins). With more and more of these fluorescent antibodies available and an increasing number of channels available in the flow cytometers, immunophenotyping is becoming more complex over time.
The ectopic expression of foreign proteins is a popular tool to study cell signaling pathways and the function of specific signaling molecules. Flow cytometry is probably the easiest and fastest way to assess the efficiency of transfection (or transduction).
Using fluorescent dyes that are only able to penetrate cell membranes of apoptotic or pre-apoptotic cells, the detection of dying cells within a population of cells is very easy. Available dyes for apoptosis detection include Annexin V or 7ADD.
Cell Cycle Analysis
Using fluorescent probes that bind to DNA, one can analyze the amount of DNA in each cell and correlate the relative amount of DNA within the cell population to a specific point of the cell cycle. Popular dyes for cell cycle analysis include propidium iodide, DAPI or ethidium bromide.
Using freely diffusing fluorescent dyes like carboxyfluorescein succinimidyl ester (CFSE) allows for the homogenous staining of a whole cell population. The staining is passed on to the daughter cells and by calculating the dilution of CFSE, one can extrapolate the proliferation of cell samples over time.
Nowadays, cell proliferation can also be analyzed with photoswitchable dyes. One example is Kikume, a green-to-red switchable fluorescent protein. The idea is to photoswitch a fluorescent protein at timepoint zero and observe two things over time.
A) The step by step decrease of the photoswitched fluorescent protein.
B) The step by step increase of newly synthesized fluorescent protein.
Looking at ratios of A and B, the proliferation of cells can be analyzed in much more depth.
This has successfully been done by Elena Seiß, part of our pxONE user family.
Intracellular increase of calcium concentration is a very common early response to external stimulation of many different cell types. Calcium enters the cytoplasm from intracellular calcium stores in the endoplasmic reticulum (ER) as well as from the extracellular space through membrane bound calcium ion channels.
Calcium dyes bind calcium ions and change their fluorescent properties/intensity. This change is detected by the cytometer over time and allows the generation of kinetic calcium curves. There are mainly two different kind of calcium dyes. Non-ratiometric calcium dyes, like Fluo-3, increase their fluorescent intensity when bound to calcium. The increase of fluorescence can be correlated to the amount of calcium that entered the cell over time. Ratiometric dyes, like Indo-1 change their fluorescent properties when bound to calcium. “Free” Indo-1 emits at ~530 nm and binding to calcium shifts the emission to ~390 nm. The calculation of relative ratios of bound versus unbound Indo-1 allows for a much more sensitive detection of changes in calcium concentration.
The main advantages of using flow cytometry for calcium analysis are the increased signal sensitivity and much higher throughput compared to fluorescence microscopy. Furthermore, the multiparameter analysis allows to run internal negative controls or compare the calcium flux ability of several subpopulations within one cell sample in one experiment.
FRET (Fluorescence Resonance Energy Transfer)
FRET is a method to analyze the proximity between two fluorescent proteins. The basic idea is that the wavelength of the emitted light from fluorophore A is similar to the wavelength required for excitation of fluorophore B. When fluorophore A and B are in close proximity, excitation of fluorophore A (FRET donor) will lead to light emission from fluorophore B (FRET acceptor). The fusion of fluorophores A and B to two different signaling molecules allows the dynamic analysis of proximity between these two proteins.
FRET can increase the resolution of flow cytometry by indirectly assessing the localization of a protein. For example, one can fuse a red fluorescent protein to the plasma membrane and monitor the recruitment of a GFP-tagged protein to the membrane by detecting a FRET signal. This becomes even more interesting when using photoswitchable dyes, where the donor and/or acceptor of the FRET pair can be switched on or off using light. The combination of flow, FRET and photoswitchable dyes is a powerful tool to study signaling pathways and dynamic relocalization of signaling molecules.
The combination of flow, optogenetics and FRET has successfully been established by our pxONE user family member Frederic Larbret.
Biosensors are bioengineered detection systems that can be expressed in cellular systems and are used to monitor specific signaling processes inside a cell. Usually, the concept behind biosensors is that the sensor can bind to a cellular component of interest (e.g. second messenger) which elicits a conformational change leading to a reporter signal that can be either fluorescent or luminescent.
One popular example is the biosensor GECO which increases its fluorescent intensity in the presence of calcium.
Fluorescence Activated Cell Sorting (FACS)
A FACS machine, or cell sorter, is very similar to a classical flow cytometer. In addition to analyzing cells that travel inside a capillary through laser beams, a cell sorter can separate single cells from within the cell sample to either a tube, or various cell culture plates right after the analysis.
Cell sorting can be used to generate cell lines, enrich transfected or transduced cells, or for further analysis (e.g. single cell sequencing).
Flow Cytometry and Optogenetics
Until recently, the combination of flow and optogenetics, or flow with any photo-regulated cell sample, was impossible. Most people were and still are using fluorescent microscopy. This makes perfect sense for all assays that require subcellular resolution. But for all other assays, flow offers a high-content, high-throughput alternative. Moreover, there are no limitations to the choice of fluorophore. The photostimulation happens in a different place and time from the analysis, so you can use the whole spectrum of fluorophores for your experiments.
LED Illumination for Optogenetic Flow Cytometry
Combining flow cytometry with precise photo-regulation of your cell sample has never been this easy. We at opto biolabs develop illumination devices suited to your experimental approach. Have a look in our product section and find the right illumination device for your flow cytometer. Do not hesitate to contact us if you cannot find a suitable solution. We build customized illumination devices tailored to your problem.
Check out our pxONE here and get ready for your optogenetic flow cytometry experiments.