How Flow Cytometry Works
Flow cytometry measures physical and fluorescent properties of individual cells in suspension. It processes thousands of cells per second, giving you single-cell resolution at high throughput. This page explains the core components and principles of the technique.
This short video gives a quick visual overview of how flow cytometry works:
The Instrument
Section titled “The Instrument”A flow cytometer has three systems: fluidics, optics, and electronics. Each plays a distinct role in turning a tube of cells into measurable data.
Fluidics: Getting Cells in a Single Line
Section titled “Fluidics: Getting Cells in a Single Line”The fluidics system delivers cells to the laser one at a time. Your cell suspension is injected into a stream of sheath fluid. The sheath fluid flows faster than the sample stream. This speed difference creates hydrodynamic focusing, which squeezes cells into a single-file line.
Why does this matter? If two cells pass the laser at the same time, their signals overlap and you get garbage data. Hydrodynamic focusing prevents this by ensuring cells arrive one by one.
Optics: Lasers and Filters
Section titled “Optics: Lasers and Filters”Lasers provide the excitation light. Common lasers include:
- Blue (488 nm): the workhorse laser, excites FITC, PE, and PerCP
- Red (633 nm): excites APC and its tandems
- Violet (405 nm): excites BV421, BV510, and other Brilliant Violet dyes
- UV (355 nm): used in high-end instruments for additional colors
When laser light hits a cell, two things happen. Some light scatters off the cell. If the cell carries fluorochrome-labeled antibodies, those fluorochromes absorb the laser light and emit light at a longer wavelength.
Optical filters separate the emitted light by wavelength. Bandpass filters let through a narrow range of wavelengths. Dichroic mirrors reflect some wavelengths and transmit others. Together, they direct each fluorochrome’s emission to the correct detector.
Detectors: Turning Photons into Numbers
Section titled “Detectors: Turning Photons into Numbers”Photomultiplier tubes (PMTs) are the most common detectors. They convert photons into electrical signals and amplify them. Some newer instruments use avalanche photodiodes, which offer improved sensitivity and a more compact design.
The electrical signal from each detector is digitized and recorded for every cell. The result is a matrix: one row per cell, one column per parameter measured.
Spectral Flow Cytometry
Section titled “Spectral Flow Cytometry”Traditional flow cytometers use bandpass filters to collect a narrow slice of each fluorochrome’s emission. Spectral flow cytometers take a different approach. They capture the full emission spectrum of every fluorochrome across all wavelengths. Software then uses spectral unmixing to separate overlapping signals. This allows more fluorochromes per experiment and better resolution of dim signals.
This video explains how spectral flow cytometry improves on conventional instrumentation:
Scatter Parameters
Section titled “Scatter Parameters”Every flow cytometer measures at least two scatter parameters without any fluorescent staining.
Forward Scatter (FSC)
Section titled “Forward Scatter (FSC)”Forward scatter measures light scattered in the forward direction, roughly along the laser beam path. It correlates with cell size. Larger cells scatter more light forward.
Side Scatter (SSC)
Section titled “Side Scatter (SSC)”Side scatter measures light scattered at 90 degrees to the laser. It correlates with internal complexity and granularity. Cells with more internal structures like granules scatter more light sideways.
Identifying Cell Types with FSC vs SSC
Section titled “Identifying Cell Types with FSC vs SSC”A simple FSC vs SSC dot plot of peripheral blood reveals three distinct populations without any antibody staining:
| Population | FSC | SSC | Reason |
|---|---|---|---|
| Lymphocytes | Low | Low | Small cells, few granules |
| Monocytes | High | Medium | Larger cells, moderate complexity |
| Granulocytes | High | High | Large cells, packed with granules |
This is often the first plot you look at. It tells you whether your sample is intact and helps you set initial gates.
Fluorescence
Section titled “Fluorescence”Scatter parameters alone cannot distinguish T cells from B cells or CD4 from CD8 T cells. For that, you need fluorescent labeling.
Antibodies and Fluorochromes
Section titled “Antibodies and Fluorochromes”The standard approach uses monoclonal antibodies conjugated to fluorochromes. Each antibody binds a specific surface protein on the cell. For example:
- Anti-CD3 antibody labels T cells
- Anti-CD19 antibody labels B cells
- Anti-CD14 antibody labels monocytes
The fluorochrome attached to each antibody determines which laser excites it and which detector collects its emission.
Excitation and Emission Spectra
Section titled “Excitation and Emission Spectra”Every fluorochrome has two spectra:
- Excitation spectrum: the range of wavelengths that can excite the fluorochrome. The laser wavelength should fall within this range.
- Emission spectrum: the range of wavelengths the fluorochrome emits after excitation. The emission is always at longer wavelengths than the excitation.
The difference between peak excitation and peak emission is called the Stokes shift. A larger Stokes shift makes it easier to separate excitation light from emission light.
Spectral Overlap
Section titled “Spectral Overlap”Fluorochrome emission spectra are broad, not narrow spikes. This means the emission from one fluorochrome can bleed into the detector meant for another fluorochrome. This is called spectral overlap or spillover.
For example, FITC emits primarily at 519 nm but also emits significant light at 575 nm, which is the PE detector range. If you do not correct for this, a FITC-bright cell looks like it is also PE-positive when it is not.
Correcting for this spillover is called compensation. It is critical for accurate multi-color flow cytometry and is covered in detail on the next page.
Common Applications
Section titled “Common Applications”Flow cytometry is used across immunology, cell biology, and clinical diagnostics.
Immunophenotyping
Section titled “Immunophenotyping”The most common application. You stain cells with a panel of fluorochrome-conjugated antibodies to identify and quantify immune cell populations. Clinical examples include CD4 T cell counting in HIV monitoring and leukemia/lymphoma diagnosis.
Cell Cycle Analysis
Section titled “Cell Cycle Analysis”DNA-binding dyes like propidium iodide or DAPI stain cells proportionally to their DNA content. Cells in G1 have 2N DNA. Cells in S phase have between 2N and 4N. Cells in G2/M have 4N. The resulting histogram shows distinct peaks for each phase.
Apoptosis
Section titled “Apoptosis”Annexin V binds phosphatidylserine, which flips to the outer leaflet of the cell membrane during early apoptosis. Combined with a viability dye, you can distinguish live cells, early apoptotic cells, and late apoptotic or necrotic cells.
Intracellular Cytokine Staining
Section titled “Intracellular Cytokine Staining”After stimulating cells with an antigen and blocking secretion with brefeldin A, you can fix and permeabilize cells to stain intracellular cytokines. This reveals which cells produce specific cytokines like IFN-gamma or TNF-alpha.
Cell Sorting (FACS)
Section titled “Cell Sorting (FACS)”Fluorescence-Activated Cell Sorting is a specialized application where the instrument physically separates cells based on their fluorescence properties. After laser interrogation, the stream is broken into droplets. Droplets containing target cells receive an electrical charge and are deflected into collection tubes.
Sorted cells are viable and can be used for downstream experiments like culture, sequencing, or transplantation.
Key Terms
Section titled “Key Terms”| Term | Definition |
|---|---|
| Hydrodynamic focusing | Sheath fluid forces cells into a single-file stream |
| FSC | Forward scatter, correlates with cell size |
| SSC | Side scatter, correlates with internal complexity |
| Fluorochrome | A molecule that absorbs light at one wavelength and emits at a longer wavelength |
| Spectral overlap | Emission from one fluorochrome bleeding into another detector channel |
| Compensation | Mathematical correction for spectral overlap |
| PMT | Photomultiplier tube, the detector that converts photons to electrical signals |
| FACS | Fluorescence-Activated Cell Sorting, physically separates cells by fluorescence |
| FMO | Fluorescence Minus One, a control used to set gate boundaries |
Summary
Section titled “Summary”A flow cytometer uses hydrodynamic focusing to pass cells one at a time through laser beams. Scatter measurements reveal cell size and granularity. Fluorochrome-conjugated antibodies enable identification of specific cell types and functional states. The combination of high throughput and single-cell resolution makes flow cytometry a foundational tool in immunology and cell biology.
The next page covers compensation and panel design, which are essential for running multi-color experiments accurately.