Biology Reference
In-Depth Information
Thus, although the capture of the emitted fluorescence may be set up with
di
erent light paths in 2P microsscopes compared to confocal single-photon
microscopes, it is conceptually similar in that emission light needs to be split by
the excitation light with the insertion of an appropriate dichroic mirror. However,
in the case of 2P microscopy, the emitted fluorescence will in most cases have a very
much shorter wavelength than the excitation wavelength, which is in contrast to
confocal single-photon and conventional epifluorescence microscopes.
Because of the abovementioned properties of 2P excitation and di
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erences to
single-photon confocal excitation, 2P excitation o
ers several important advan-
tages over single-photon excitation. First, because light scattering declines steeply
with increasing wavelengths, red and IR (670-1100 nm) light can penetrate and
hence excite fluorescent molecules much deeper into the specimen than single-
photon lasers providing excitation wavelengths in the range from
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300 to
cient power outputs and optimized optics including corrective
optics for pulse dispersion, penetration depths approaching 1000 m m (1 mm) may
be achieved, which is considerably deeper than the
600 nm. With su
Y
40-50 m m achievable with
single-photon excitation. Thus, 2P excitation secures an up to
20-fold deeper
penetration depths by using excitation light of twice the wavelength. To achieve
comparable deep imaging with single-photon confocal microscopy, tissue or layer
removal with histologic techniques or penetration by the objective would have been
required. Such mechanical approaches would for obvious reasons compromise the
''intactness'' and viability of the tissue. For this reason, confocal microscopy is
mainly restricted to studies of single, isolated cells or tissue surfaces, whereas 2P
excitation laser scanning microscopy allows deep tissue imaging of intact organs.
Similarly, 2P excitation microscopes are comparably more often upright rather
than inverted, to allow for tissue preparation to be set up on the stage, whereupon
the objective lens is lowered down to within the optical range for imaging. The
opposite is true for confocal microscopes, which more often than not come
inverted. Furthermore, even with 2P excitation deep tissue imaging, one can be
assured that the vast majority of the fluorescence comes from the focal point and
not from residual scattering (scattering is greatly reduced, but not obliterated, by
long-wavelength excitation). This is because, even in strongly scattering tissue such
as the heart, the density of scattering exciting photons is too low to generate
significant fluorescence. Therefore, this together with the long excitation wave-
lengths also contributes to 2P excitation microscopes being much less sensitive to
light scattering than regular widefield epifluorescence or confocal microscopes.
However, it should be noted that penetration depths also heavily depend on the
specimen, as di
erent tissue properties may degrade the illumination as well as the
emitted fluorescent light. In particular, collagen and myelin are known to scatter
light and thereby restrict penetration ( Helmchen and Denk, 2005 ).
Additionally, IR light is much less phototoxic than shorter wavelengths, such
that it correspondingly may cause only negligible photodamage. Also, because of
the nonlinear excitation, photodamage and bleaching are restricted to the focal
point. Importantly, the reduced photodamage and fluorophore bleaching allows
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