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500 nm (though RH-237 would not be optimally excited by this wavelength), but
have emission spectra that may be spectrally di
V
erentiated, as Fluo-3 has a narrow
emission spectrum that peaks at
660 nm
with a broad spectrum (
Fig. 11
C). Thus, this combination appears attractive as it
di
525 nm, whereas RH-237 peaks at
erentiates the Ca
2
þ
and membrane potential signals, for which it has also been
used successfully (
Fast and Ideker, 2000
).
However, several problems arise when transferring from single-photon to 2P
excitation, although several of the voltage-sensitive dyes present with consistent
and reproducible 2P excitation spectra that very much resemble the double single-
photon excitation spectra and may thus be confidently used for meaningful multi-
photon imaging. In contrast, the 2P behavior of many of the Ca
2
þ
-sensitive dyes is
somewhat di
V
cult to interpret (see previous discussion and
Fig. 8
), although the
ratiometric Fura dyes may be 2P excited in order to provide a meaningful Ca
2
þ
signal that also captures transient changes over a millisecond scale with high
fidelity (
Wokosin et al., 2004
). This may be because the Fura dyes have a single-
photon excitation spectrum in the UV range (340-380 nm), and therefore the 2P
excitation spectrum, which approximately is double the single-photon spectrum,
occurs at
Y
800 nm wavelengths, in which the 2P laser power outputs are not
limited. Moreover, this study indicated that several of the Fura dyes, in particular
Fura-4F, may work well when excited with a single IR wavelength, despite their
use as ratiometric single-photon dyes, as judged by the dynamic ranges and SNR
obtained during 2P excitation microscopy in single cardiac muscle cells during
di
erent Ca
2
þ
conditions. In contrast, the Fluo- and Rhod-based Ca
2
þ
-sensitive
dyes, all with single-photon excitation peaks at
V
500-550 nm, present with 2P
excitation spectra that are not immediately predicted by the doubled single-photon
spectra (
Fig. 8
). In these cases, the 2P excitation spectra are at least partly broken
up and appear blueshifted compared to the doubled single-photon spectra.
Although doubling the single-photon excitation spectrum is often a good predictor
for the 2P excitation spectrum, deviations from this do occur, although these
deviations may neither be systematic nor well understood (
Xu et al., 1996; Zipfel
et al., 2003
). Alongside this, a reoccurring problem is that the available Ti:Sapphire
pulsed 2P lasers are power-limited at the long wavelengths of 1000-1100 nm that
would correspond to the doubled single-photon excitation spectra of Fluo-3 and
Rhod-2. Because not all fluorophores are easily transferable from single-photon
excitation, for which they were developed, to 2P excitation, this therefore has made
it problematic to use multiple fluorophores simultaneously during 2P excitation
microscopy, and the issue has not yet been fully resolved.
Adi
erent approach to capture more complex information has been to combine
several multimodal microscopy techniques in ways that also encompass confocal and
multiphoton systems, but also this comes with both advantages and disadvantages.
For instance, di
V
erent modes of contrast used on the same specimenmay increase the
information extracted from the images and reduce artifacts. Multimodal microscopy
may also allow for a wider repertoire of fluorophores. However, if confocal and
multiphoton imaging are combined,
V
it requires descanning and insertion of a
