Flow Cytometry | Cell counting | Cell Sorting | Scattering | Basic Science Series
Flow Cytometry | Cell counting | Cell Sorting | Scattering | Basic Science Series
Flow Cytometry is a technique used to detect and measure the physical and chemical characteristics of a population of cells.
Flow cytometry is a technique used to detect and measure the physical and chemical characteristics of a population of cells.
A sample containing cells is suspended in a fluid and injected into the flow cytometer instrument.
The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components.
Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths.
Tens of thousands of cells can be quickly examined and the data gathered are processed by computer software.
Data is mainly collected in the form of forward scattered light and side scattered light measurements.
The typical plot acquired using flow cytometer looks like this wherein x-axis is the forward scattered measurements and in y-axis you have side scattered measurements. Each dot represents the individual cell or particle.
I have plotted the data using Matlab but you can also get the plot using in-build software packages with the instrument.
A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing.
The measuring system commonly uses the measurement of impedance (or conductivity). optical systems include lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers argon (488 nm), red-HeNe (633 nm), green-HeNe; diode lasers (blue, green, red, violet) resulting in light signals. The detector and analog-to-digital conversion (ADC) system convert analog measurements into digital signals that can be processed by a computer.
The amplification system can be linear or logarithmic.
The process of collecting data from samples using the flow cytometer is termed 'acquisition'. The acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer.
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