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confocal aperture with a pinhole into the light pathway, which may reduce the
fluorescence capture after 2P excitation and thus lead to a loss of signal, though a
confocal aperture may also be set up to increase the spatial resolution of multiphoton
images, by restricting the PSF tails. Because of these limitations, the reality is often
that it is di
Y
cult, though not impossible, to achieve optimal performance from each
individual mode when several modes are combined. Nonetheless, the advantages of
simultaneous or near-simultaneous light capture by di
erent modes of microscopy
may, under the right circumstances, far outweigh the disadvantages.
Examples include combinations of confocal or multiphoton with epifluorescence
or di
V
erential interference contrast microscopes to capture light emission restricted
to the focal plane as well as capturing a widefield view, either simultaneously or
sequentially without having to reorient or replace the specimen. Other options also
include setting up a microscope system that combines confocal and 2P excitation
imaging modes, or 2P excitation and second-harmonic generation (SHG) imaging.
Although these applications tend to serve narrow and specific purposes, they may
allow for imaging of local versus global Ca 2 þ signaling, or Ca 2 þ signaling in
combination with for example, metabolic parameters by using 2P excitation to
excite metabolites such as NADH and FAD, or collagen that in particular con-
tributes to the SHG signal ( Masters, 2006 ). A final example of multimodal micros-
copy techniques that may successfully be combined includes the combination of
FRET and FLIM imaging to quantify FRET between two fluorophores, as in the
case of the Ca 2 þ -sensitive cameleon described above. These examples are not
exhaustive, but serve to illustrate the potential of combining di
V
erent fluorophores
or microscopy modalities in order to gain information of a more detailed nature.
V
References
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