Flow Cytometry Core Service


Background

Flow cytometry is a technique used for measuring and analysing certain characteristics of a single particle or cell. The particles or cells move through a fluid system and pass through a laser where the relative size, granularity / internal complexity and the fluorescence intensity associated with the particle are measured. A Flow Cytometer comprises a fluidics, optics and electronics systems.


Optics

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A series of prisms and lenses guides laser light to the cuvette where it intercepts the hydrodynamically focused sample stream. At this point any fluorescence associated with the particle and scatter information of the particle can be obtained.

Light scattered in the forward direction is collected by the Forward Scatter channel (FSC) photodiode and provides information on the relative size of the particle. Light measured at a 90° angle to the laser excitation beam is collected by the Side Scatter channel (SSC) photomultiplier tube (PMT) and provides information on the relative granularity of the particle.

Separate fluorescence channels (FL-) detect any fluorescent light associated with the particle. The light is directed via a series of mirrors and filters to PMT´s. Filters determine the wavelength of light to be detected by the PMT, any unfiltered light is reflected by a dichoric filter and is channelled to the next fluorescent channel.

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Fluidics System

The fluidics system is essential for transporting the particles from a random mix into and orderly stream of single file particles.

The point at which the particles are delivered into the sheath fluid is commonly termed the Flow Cell. The sample is injected into the core of the sheath fluid stream, which then accelerate and pass through a narrowing channel, channelling the particles in the core of the sheath fluid into single file. This phenomenon is known as hydrodynamic focusing and presents a single file of particles to the laser/s.


Electronics

Light hits the photodetector and a small current is generated, the associated voltage of which is amplified and then represented by electrical signals via an analogue to digital converter. These electrical signals are what are seen on the computer connected to the Cytometer.