Biology Reference
In-Depth Information
Figure 5.1 Fluorescence explained: Jablonski diagram. Source: Dr Thomas G. Chasteen
at Sam Houston State University, Huntsville, Texas. (For color version of this figure, the
reader is referred to the online version of this topic.)
states eventually leads to molecular damage with a gradual reduction of flu-
orescence emission intensity from a sample over time. This limits the length
of time for which a sample can be observed. Many fluorophore labels are
conjugated to antibodies and therefore the specificity depends upon that of
the antibody. Often, it is not possible to distinguish between different spe-
cies, especially for protozoa where the cell membrane structure is generally
highly conserved between species.
For Cryptosporidium detection, the fact that the antibody binds all spe-
cies is advantageous since the test thus determines whether any oocysts are
present. The disadvantage is that no information is gained about the human
pathogenicity of detected oocysts. Another problem is that this antibody
displays some cross-reactivity to algal cells, which are of a similar size to
oocysts, and this can complicate detection. Antibodies are discussed further
in Chapter 7, as they are often utilized in biosensors to capture the pathogen
of interest.
Fluorophores can also be conjugated to a wide variety of other mol-
ecules or incorporated into detection schemes where interaction with other
molecules controls the production of fluorescence ( Fig. 5.2 ). We have seen
above how antibody-based fluorophores can be utilized to bind to anti-
gens on the cell surface. Alternatively, chemicals such as 4′,6-diamidino-
2-phenylindole (DAPI) can be employed which become fluorescent upon
interaction with nucleic acids. DAPI can therefore be used for nonspecific
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