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for longer imaging periods. 2P excitation spectra of most fluorophores are wider
than the equivalent single-photon excitation spectra, whereas the associated emis-
sion spectra after 2P and single-photon excitation are not di
erent, such that the
same excitation wavelength may potentially excite several fluorophores with dis-
tinct emission spectra ( Bestvater et al., 2002; Xu et al., 1996 ). This property will be
explored in more detail later in the chapter. Moreover, whereas the positioning of
the confocal aperture relative to the light detector as well as the focal plane is
critical to ensure that the images of the source and detector apertures are cofo-
cused, this is not the case for 2P excitation microscopes, in which the position of
the light detector is not critical, because fluorescence emission will only be gener-
ated at and tightly around the focal volume.
However, several problems persist with 2P excitation. First, 2P excitation requires
very high light intensities; intensities that would instantly vaporize the specimen if
the light was delivered continuously. However, this is overcome by using lasers that
provide ultrabrief (10 13 s) pulses at very high frequencies (
V
80-100 MHz), which
thereby generate very high instantaneous energy, but su
ciently low average energy
to avoid any substantial damage to the specimen. Thus, the distance between each
pulse is typically
Y
10 ns, whereas the width of each pulse itself is typically 100 fs
( Fig. 6 ). The high repetition rates match fluorescence lifetimes closely (see above)
such that a good balance between excitation e
ciency and onset of saturation is
achieved ( Helmchen and Denk, 2005 ). The most widely used lasers that fulfill this
criterion and provide the necessary wavelengths, are the solid state titanium:sap-
phire (Ti:Sapphire) oscillating (pulsed) lasers. These lasers are tunable, such that the
latest versions are capable of delivering wavelengths within the range
Y
670-
1100 nm, thus, from visible red to infrared (IR), though this range is continuously
expanding while also coming with higher power outputs, as manufacturers keep
developing their product lines. Though most 2P excitation applications may not be
power-limited, more available power does benefit applications requiring deep
tissue penetration. Typical power outputs in the latest Ti:Sapphire lasers may exceed
4 W at 800 nm, though the optics cause a major loss in power from the outlet of the
laser to the focal point of the objective lens. Glass components of the optical
pathway also cause a dispersion of the
100 fs laser pulses, but this may be
compensated for by corrective optics in order to maximize 2P excitation ( Diels
et al., 1985 ). Other limitations of 2P excitation microscopy are that reflected light
imaging is not possible; only fluorescence imaging, and that it is not suitable for
imaging highly pigmented specimens, as these absorb IR and near-IR light.
XII. Ca 2 þ Indicators for Use in Confocal and
Multiphoton Microscopy
For many years, biology has benefited from the fluorescent dyes or constructs
that bind and therefore can measure the free concentration of Ca 2 þ [Ca 2 þ ]. These
indicators have been used to examine the levels and time course of changes
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