Flow Cytometry

Topics: Light, Flow cytometry, Cell biology Pages: 5 (1635 words) Published: April 28, 2013
Cytometry is a combination of two greek words: cyto- cell, and metry - measurement. So cyto-metry is about making cell measurements. To elaborate, flow cytometry is a technique for analysing particles (cells in this case) which are suspended in a fluid stream by flowing the cells past an interrogation point. A light source is directed on to a hydrodynamically focused stream to create a zone for interrogation. As cells flow through this interrogation zone they scatter the light, at this point any fluorescent compounds that are inside or attached to the cell can be excited by the light source and emit light themselves, at a longer wavelength to the excitation source. Multiparametric information about the physical and chemical characteristics of a cell can be determined from the detection and analysis of both the scattered and fluorescent light.

There are 3 key components in a flow cytometer, which include Fluidics, Optics and Electronics. The fluidics deals with delivering the sample to the interrogation point one cell at a time. The sheath fluid and the sample are pressurised and used to deliver the sample to the interrogation point. One of the basic principles of flow cytometry is the ability to analyze individual particles, or cells. The main purpose of the fluidics system is to deliver the sample to the interrogation zone one cell at a time. When a sample is taken into a flow cytometer the particles are randomly dispersed throughout the suspending medium, the fluidics system is able to align this randomly dispersed sample to a precise, single cell stream. Central to the fluidics system is the flow chamber, there are two types of flow chambers; jet-in-air and in cuvette. The flow chamber consists of a core channel through which the sample is injected under pressure. Surrounding this core channel is an outer chamber with flowing sheath fluid which, due to the narrowing dimensions of the flow chamber flows faster than the injected sample. This fast flowing sheath produces a huge drag effect on the core chamber, this drag leads to the dispersed sample to be pulled into alignment; this effect is known as hydrodynamic focusing. Once aligned the sheath and the sample are forced parallel out of the flow chamber under laminar flow conditions ready to be interrogated as they flow past the light source.

In laminar flow the sample and sheath do not actually mix. The difference between the sample and sheath pressure is known as the sample differential. The greater the sample differential the faster the sample will flow in to the flow chamber, this will increase the event rate, or number of cells interrogated in any given time. The speed at which the sample is forced into the flow chamber is known as the flow rate. How well aligned the cells are when delivered to the interrogation zone depends on the flow rate.

In a flow cytometer, the light source is typically a laser or an arc lamp. Lasers can provide a single wavelength of coherent light at one or multiple discrete frequencies; in contrast arc lamps use the light emissions of gases that are ionized by a high voltage. This produces non-coherent light of multiple wavelengths so needs subsequent optical filtering before being focused on the stream. Lenses can be used to shape and focus the excitation light, and to direct the excitation light to the stream. The preamble of clean-up filters, lenses, mirrors, light source, are all part of the excitation optics. Where the laser intersects the stream is the interrogation zone, as a particle passes through this zone there is light scatter and possibly fluorescence; it is the detection and analysis of this light scatter and fluorescence that gives information about a cell. In order to detect the light scatter and fluorescent light there are detectors; typically one in line with the laser (to detect forward scatter) and several collecting light perpendicular to the laser - this is also where side...
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