of assessment of immunocytochemical staining using conventional microscopy
is that it is very difficult to quantify the intensity of the immunostaining.
Although it is possible to estimate the proportion
s that are immunostained in a population
by performing manual
cell counts, the accuracy
of the estimate is dependant on the total number of cells that are counted and the proportion of positive cells.
are overcome in flow cytofluorimetric analysis. In this technique
, the measurement of the size
and refractive index of individual cells in a fluid stream
These cells intersect a laser beam and reflected light is collected by a photomultiplier tube set at right angles, hence "side scatter." Refracted light around the cell is proportional
to size and is collected incident to the laser beam, hence "forward scatter."
If the cells are incubated with fluorescent proteins, usually an antibody
against a specific cell surface antigen
this can be excited by the high energy laser and the Stokes-shift emitted light is collected.
Up to three wavelengths can normally be detected, allowing cellular discrimination according to five parameter
s. The collected light from each cell is converted into electron
data which is processed by the computer and can be presented as coordinates on a dot plot or as a historgram on screen.