resulting fluorescence. If europium is used the stimulatory wavelength is 340 nm,
and the fluorescence generated is 615 nm.7.4 Immuno microscopy
7.4.1 Immunofluorescent (IF) microscopy
Immunofluorescent (IF) microscopy uses antibodies conjugated to fluorescent markers
to locate specific structures on specimens and allows them to be visualised by
illuminating them with ultraviolet light.Fluorosceinandrhodamineare the usual
labels used but alternative markers are available. Fluoroscein produces a green
fluorescence and rhodamine is red. Microscopes equipped to carry out IF have dual
sources of light allowing the operator to view the specimen under white light before
illuminating with ultraviolet to look for specific fluorescence. The technique is
particularly useful for looking at surface markers on eukaryotic cells but is also used
as a whole-cell staining technique in bacteriology.
Membrane studies on whole mammalian cells can be undertaken and themigration,
endocytosis(uptake of membrane-bound particles by cells) and fate of labelled
receptors studied in real time. Bound receptors in cell membranes frequently migrate
to one end of the cell prior to being endocytosed. This phenomenon is known as
cappingand is easily viewed in living cells using antibodies specific to cell membrane
receptors labelled with a fluorescent marker.7.4.2 Immunosorbent electron microscopy
Immunosorbent electron microscopy(ISEM) is a diagnostic technique used primarily
in virology. Virus-specific antibodies conjugated togold particlesare used to visualise
virus particles on electron microscopes. The gold is electron-dense and is seen as a
dark shadow against the light background of the specimen field. The technique can be
used for both transmission or scanning systems. If gold-labelled primary antibodies
are not available then anti-IgG–gold conjugated antibodies can be used with the
primary antibody in a double antibody system. Both monoclonal and polyclonal
antibodies can be used for ISEM depending on the required specificity.7.5 Lateral flow devices
Lateral flow devices(LFD) are used as rapid diagnostic platforms allowing almost
instant results from fluid samples (Fig. 7.16). They are simple to use and contain all of
the required components within the strip itself. They are usually supplied as a plastic
cassette with a port for applying the sample and an observation window for viewing
the result. The technology is based on a solid phase consisting of a nitrocellulose or
polycarbonate membrane which has a detection zone which is coated with a trapping291 7.5 Lateral flow devices