Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 88

pixel location [ 102 - 105 ]. This is an ideal technique to implement FRET imaging
in living cells and to establish the existence of a FRET signal “on the fly.” Given
the reference spectra obtained from the donor-alone and acceptor-alone specimens,
linear unmixing the œ-stack acquired from a specimen containing both donors
and acceptors can separate the signals emitted from the donors and the acceptors.
Thus, spectral imaging microscopy combined with linear unmixing provides an
accurate way of removing the SBT contaminations resulting from the donor [ 103 ].
In sFRET microscopy, the acceptor SBT is removed by employing the same strategy
used in the PFRET approach. Recently, we have developed a straightforward
3-color spectral FRET (3sFRET) microscopy method and demonstrated its utility
for studying the interactions between three fluorescently labeled proteins in a single
living cell [ 43 , 106 ]. The 3-color FRET microscopy approach provides the capability
to simultaneously track the interactions between three fluorescently labeled cellular
components that form complexes, clusters, or discrete associations during cellular
signaling or trafficking events in the same region of interest over four dimensions
(3-D C time).
To evaluate the accuracies of different FRET microscopy methods, the Vogel
laboratory (NIH/NIAAA) developed a set of genetic constructs encoding fusion
proteins containing donor and acceptor FPs separated by protein linkers of defined
length [ 96 , 107 ]. For each genetic construct expressed in living cells, there was
consensus in the E results obtained by different FRET microscopy methods
[ 100 , 107 ], demonstrating that they could serve as FRET standards. The FRET-
standard approach has been used by us to calibrate our FRET microscopy methods
(see an example in Sect. 3.4.3 ) as well as to test the utilities of new FPs for FRET
studies [ 47 , 108 ].

3.3.5 Other Advanced Microscopy Techniques

Total internal reflection fluorescence (TIRF) microscopy provides a restricted
illumination depth (typically < 100nm) into a specimen using an evanescent wave
which is generated by having the excitation light totally reflected at the surface
between the glass coverslip (refractive index D 1:518) and the aqueous medium
of the specimen (refractive index D 1:33 1:37). Thus, TIRF microscopy yields an
ultrahigh signal-to-noise ratio for imaging fluorescent molecules within 100 nm
above the coverslip and is an ideal live-cell imaging tool for visualizing membrane
proteins, studying protein-protein interactions at cell membrane surface, and inves-
tigating the mechanism and dynamics of many proteins involved in cell-cell contact
[ 109 - 111 ]. TIRF microscopy has also been used for imaging single molecules and
superresolution microscopy imaging (described below). TIRF microscopy can be
purchased from any leading commercial microscope companies such as Carl Zeiss,
Leica, Nikon, Olympus, etc.
Fluorescence recovery after photobleaching (FRAP) microscopy and associated
techniques (e.g., fluorescence loss in photobleaching (FLIP) microscopy) are very