Flow cytometry involves the analysis of the fluorescence and light scatter properties of single particles (e.g. cells, nuclei, chromosomes) during their passage within a narrow, precisely defined liquid stream. Figufe 1. Close up of the flow chamber. A typical flow cytometer consists of several basic components: a light source, a flow chamber and optical assembly, photodetectors and processors to convert light signals into analog electrical impulses, analog-to-digital converters, and a computer system for analysis and storage of digitized data. Figure 2. Schematic view of a flow cytometer and sorter with a jet-in-air configuration showing one forward light scatter and two fluorescence detectors. The particles in the flow stream scatter the illuminating light. Simultaneously, if particles have previously been stained with a fluorescent dye capable of absorbing the illuminating light, fluorescence emission will occur. Some particles may contain natural fluorochromes (e.g. chlorophyll) which upon excitation also fluoresce. Scattered light and emitted fluorescence is collected by lens behind which optical filters may be placed. These are used to exclude the excitation wavelength for fluorescence measurement and, if needed, to divide the fluorescence emission for simultaneous measurement of two or more fluorescent dyes. In addition to the jet-in-air configuration described above, other flow configurations also have been developed and are used in some commercial instruments. Their advantage is that they can be operated at flow velocities lower than those of the jet-in-air configuration, which may facilitate higher sensitivity. Figure 3. Flow chamber designs as used in some commercially available flow cytometers. (A) Jet-in-air configuration; particles are measured in a narrow liquid stream after exiting the nozzle. (B, C) Enclosed-stream configurations; particles are measured while flowing in a narrow channel. (D) Jet-on-open surface configuration; particles are measured in a liquid jet while it is flowing on the glass surface. An attractive feature of flow cytometry is that flow sorters are able to separate particles of interest from heterogeneous populations. A jet-in-air configuration can be modified to break the stream into droplets which are formed by the action of a vibrating transducer attached to the flow chamber. Electronic circuitry then places an electrical charge on the fluid stream at the time when the desired particle is entering the last attached droplet. Charged droplets are deflected electrostatically by passage through an electrical field (see Fig. 2). While cytometers based on the jet-on-open surface design cannot be modified for sorting, in some enclosed-stream cytometers, sorting is made possible by switching the flow between two outlets of the flow chamber.
Flow chamber (nozzle) is a heart of a flow cytometer. The suspension of a single particles emerges from the sample needle into a surrounding steath fluid liquid that is moving with a greater velocity. The resulting acceleration at the orifice forces the particles to travel one by one in the central portion of the fluid jet that emerges from the flow chamber. This proces is called hydrodynamic focusing. Typical orifice diameters are in the range 50 - 100 µm, resulting in jet velocities between 1 -10 m/s. the particles within the stream then traverse the focus of an intense beam of light at rates in the range 100 -1000 particles/s.
Detectors (mostly photomultiplier tubes) convert light pulses to electric current pulses, which are then amplified by a linear or logarithmic amplifier. After amplification, the electronic signal is digitized for further computer processing and storage. The results of analysis are usually displayed in the form of a histogram of fluorescence intensity among the particles in the sample.1. Flow Chamber Designs
2. Particle Sorting
