Understanding the instrumentation basics
Understand how both a flow cytometer and multi-color flow cytometer work to provide detection of different fluorescent wavelengths.
The flow cytometer
When running a cell suspension through a flow cytometer, sheath fluid hydrodynamically focuses cells to get them to pass in a single file through a small nozzle. The resulting tiny stream of fluid takes cells one at a time past a laser light, as shown in Figure 1.1
Figure 1. Diagram showing an overview of the flow cytometer. Sheath fluid focuses the cell suspension, causing cells to pass through a laser beam one at a time. Forward and side-scattered light is detected, with fluorescence emitted from stained cells.
Figure 2. Overview of basic multicolor flow cytometry technology
- When a sample is introduced into the multicolor flow cytometer flow chamber, it enters the fluidics system and separates into single cells in a process known as hydrodynamic focusing. Hydrodynamic focusing uses a controlled fluid flow to focus the sample into a narrow diameter, causing the cells to separate and align in a single file.
- As each cell passes the laser, the instrument records it as an event. For each event, forward scatter (FS) and side scatter (SS) are subsequently recorded. If a cell is fluorescently labeled, the laser excites the fluorophore, and the emitted light is collected as fluorescence intensity.
- For the instrument to detect the specific wavelength emitted by a fluorophore, the emitted light is passed through a series of mirrors and filters until it reaches the appropriate detector. Detectors are known as photomultiplier tubes (PMTs) and will only detect fluorescence at a specific wavelength.
- Optical filters block certain wavelengths and let others pass. When placed at an angle, a dichroic filter acts as a mirror, allowing specific wavelengths to pass through while reflecting others. The type and order of dichroic filters allow the simultaneous detection of multiple signals.