Biomedical Engineering Reference
In-Depth Information
epifluorescence, the excitation light beam shines directly on the sample
at a 90° angle. In TIR, the light is focused through a prism into the glass
coverslip so that the sample is struck by the beam at a shallow angel
(23°). Consequently, only fluorophores near the plasma membrane are
excited by the light, rather than the bulk of the cellular material. The rest
is physics and math. However, TIR is the only fluorescent microscopy
method that allows some detailed insights into endocytic and exocytic
events at the cell surface.
Fluorescence resonance energy transfer (FRET)
Fret is a method that allows for the identification of proteins that in-
teract within live cells, either stably or transiently. FRET requires the
expression of two different interacting proteins in a cell that are each la-
beled with distinct fluorescent molecules that are capable of transferring
energy. In FRET, the exited fluorophore (the donor) transfers energy to
a second longer wavelength fluorophore (the acceptor). Note that the
transfer can also be considered a quenching reaction because there is
no emission of fluorescence by the donor fluorophore. As outlined in
Chapter 3, FRET is also used in real-time PCR.
In general, the proteins to be studied using FRET are genetically
modified as fusion proteins to contain one of two different green flu-
orescent protein derivatives, CFP and YFP (see Chapter 4 for more
information). FRET is highly sensitive to the distance between the two
labeled proteins, requiring that they be within 10 nm (or 100 Å) before
energy transfer can take place. The interaction of the two proteins
results in energy transfer, which is detected by computer-based soft-
ware that receives signals from digital cameras on the microscope. Be-
cause it is a microscopic method, FRET provides direct insights into
the intracellular compartment(s) in which protein-protein interactions
occur.
Fluorescence recovery after photobleaching (FRAP)
FRAP is a live cell imaging technique that is used to study the move-
ment and redistribution of fluorescent molecules. Most experiments now
take advantage of chimeric proteins with in-frame GFP or one of its vari-
ants. In a FRAP experiment, a portion of the cell is exposed to a high in-
tensity light from a laser to irreversibly photobleach fluorescent proteins
within a discrete region of the cell. The recovery of fluorescence in the
same region therefore is due to the migration of fluorescent molecules
from other areas of the cell into the photobleached region. FRAP has
been used to study protein mobility and dynamics in a number of cellular
Search WWH ::
Custom Search