<aside> đź’ˇ To Do:

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Overview of Flow Cytometry

<aside> đź’ˇ The basics of flow cytometry:

Cytometry 101 (FlowJo University)

Single cell data can be collected using flow cytometry: the measurement of cells in a flowing medium.  The tool used to make these measurements is called a cytometer.

Cytometers, with a few examples shown here, come in all shapes and sizes but they share some underlying principles. Cytometers are generally comprised of fluidics, optics, and electronics systems.

The fluidics system accepts the cell sample (colored red dots in this illustration) and transports it to the interrogation point, while forcing the cells into a single file stream though either hydrodynamic or acoustic focusing.

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The optics system is then engaged.  At the interrogation point, where the blue laser hits the red cells in the graphic, the cells pass through a laser beam or beams that excite florescent probes bound to selected cellular targets, emitting light of a different color for each unique probe.  That light is filtered into individual colors and the quantity of emitted photons is measured using photomultiplier tubes, or PMTs.

The quantitative aspect of this is important; cytometry measures relative amounts allowing for measurement of the intensity of expression instead of simply yes/no or on/off.

Finally, the electronics system is used to convert excited photons to voltage measurements to a singular number representing intensity of expression.

Lets take a closer look at how the numbers we work with are produced...

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A cell passes into a laser and begins to emit a signal detected as a voltage through the PMTs. Voltage increases until the cell is fully engaged in the laser , a maximum value will be detected, then the cell passes out of the laser and the voltage drops off

The result is a curve

To use the curve for data analysis you can integrate it, take its height or its width

in FCS files you get a
-H (height
-A (area)
-W (width)

Area is preferred as it
contains all the info
about the curve

One topic glossed over so far is how multiple phenotypes are identified.  Monoclonal antibodies tagged with the aforementioned florescent probes are used to bind specifically to the selected antigen that will reveal the phenotype under study.

Cytometry allows for the measure of up to approximately 50 different cellular aspects depending on the system. The measurement of these multiple aspects on many cells are then stored as a flow cytometric standard file, abbreviated as FCS.  FlowJo will accept FCS files from any cytometer.

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In this picture, a variety of T cells are tagged with different colour probes so they can be separately identified

What is flow cytometry?

Sample Types

Particle Types

Forward Scatter vs Side Scatter (FlowJo University)

Three Subsystems

A flow cytometer is composed of three main subsystems: fluidics, optics, and electronics