What is flow cytometry?

The flow system

Light scatter

Fluorescence measurement

Multiparametric measurement

Advantages of flow cytometry

Disadvantages of flow cytometry

Sample preparation

Applications

Data display

Safety

Further reading

Self Assessment

Introductory Contents Further reading Units 	Introduction  The flow cytometer 	Data analysis 	Immunofluorescence 	Some clinical applications  DNA and the cell cycle Other applications Cell proliferation Cell death Assignment 1 Assignment 2

Side Scatter

With laser light, the amount of light scattered to the side (perpendicular to the axis of the laser light) is detected in the side or right angle light scatter (SS, SSC or RALS).

The intensity of side scatter is proportional to the size, shape and optical homogeneity of cells (or other particles), optical homogeneity being the predominant parameter.

Side scatter tends to be more sensitive to inclusions within cells than forward scatter and can be used to distinguish granulated from non-granulated cells.

Fluorescence measurement

Fluorescence occurs when a molecule excited by light of one wavelength returns to the unexcited (ground) state by emitting light of a longer wavelength (Figure 1.3). The exciting and emitted light, being of different wavelengths, can be separated from one another using optical filters.

 











Figure 1.3. The absorption and emission of light during fluorescence

In flow cytometry, in addition to light scatter, one or more fluorescences are usually measured. Some specialised organisms (for example, marine algae) may be naturally fluorescent. For most applications, the cells are labelled with fluorescent probes. A commonly used probe is a monoclonal antibody with a fluorescent molecule attached. This type of label will be discussed in more detail in Units 4 and 5.

The fluorescent light is collected at right angles to the laser beam. Typically, an instrument might be capable of measuring four fluorescences - green, yellow, red and deep red – although some cytometers can measure 15 colours or more. The different fluorescent colours and the scattered light are selected and separated using optical filters, which will be described in Unit 2 2.

Multiparametric measurement

A flow cytometer may measure six or more parameters on each cell passing through the instrument (for example, forward and side scatter and fluorescences of four different colours). This is referred to as a multiparametric measurement.

A typical flow rate would be 1000 cells/sec, so that measurements may be made on tens of thousands of individual cells within less than one minute.

The only way in which this large amount of data can be handled and analysed is by using a powerful, well-programmed computer. The data is analysed in real time as the data is acquired. The raw data is also written on to the hard disc as a file, which is called a listed data file. Further analysis can be made off-line at a later date.

All the instrument manufacturers have agreed on a common format, the Flow Cytometry Standard format (FCS) for these listed data files so that programs can be written which will analyse data acquired on any instrument.

Advantages of flow cytometry

The advantage of any cytometric measurement (image or flow) is that it records data from single cells.

Measurements on single cells by a cytometric technique can be contrasted to a biochemical measurement, which records an average value for all cells in a sample.

For example, a cytometric measurement of estrogen receptors (ER) in a biopsy of a breast carcinoma would reveal the distribution of ER amongst the cells. If the tumour cells are labelled with an additional antibody, the measurements can be made on the tumour cells only, ignoring normal cells present. A biochemical measurement might merely reflect the difference in the number of tumour cells present in the sample. Also, it cannot distinguish between a sample containing cells, the majority of which have a low number of receptors, from one in which there are only a minority of strongly positive cells.


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