Biology Reference
In-Depth Information
XIII. Use of Dyes for Single-Photon Confocal Microscopy
In theory, all the fluorescent Ca
2
þ
indicators created for cell biology can be used
in confocal or 2P excitation modes, although in practice, only a limited number of
dyes are routinely used for a series of practical considerations. By far, the majority
of confocal applications use single-wavelength excitation and emission dyes. These
dyes have several advantages: (i) their excitation wavelength is
500 nm or longer
and therefore can use the emission from readily available Argon or Krypton-
Argon lasers, and (ii) the majority of dyes have a good dynamic range (see
below). But they also su
cult
because the signal will be a function of both the concentration of the dye (unknown
and variable) and the concentration of Ca
2
þ
and (ii) dye signal may vary with time
due to loss of the dye from the volume or due to photobleaching. All dyes
photobleach, but some dyes are more susceptible than others; in particular,
Fluo-3 and Fluo-4 are amongst those that most rapidly bleach (
Thomas et al.,
2000
). Dyes that show significant spectral shifts can be used in a single wavelength
mode, for example, Fura-based dyes can be imaged in confocal microscopy by
using a 405 nm laser. This produces an inverse reporter since fluorescence
decreases as Ca
2
þ
increases (
Wokosin et al., 2004
). Similar measurements using
Indo-based dyes are less easy because the narrow excitation spectrum means that
405 nm laser light only poorly excites the dye and Indo-based dyes appear to be
inherently more prone to photobleaching.
V
er from a series of disadvantages: (i) calibration is di
Y
XIV. Use of Dyes for 2P Excitation Microscopy
The utility of a dye for imaging Ca
2
þ
using 2P excitation does not follow directly
from the behavior of the dye in single-photon excitation. There is no guarantee
that 2P excitation will successfully excite the dye since the e
ciency of two-photon
excitation appears to be relative to the fluorophore structure (
Kim et al., 2008
).
A number of Ca-sensitive indicators have been studied over a limited range of 2P
wavelengths (
Xu et al., 1996
), some dyes (e.g., Oregon Green) have a better 2P
cross-section than the more common single wavelength dyes (Fluo-3); none
showed the expected increase in cross-section as the excitation wavelength
approached 900 nm. In contrast, the 2P cross-section of Fura-based dyes appeared
readily excitable by wavelengths approximately to double the appropriate single-
photon wavelength (
Wokosin et al., 2004
). In particular,
Y
800 nm light provides
an inverse Ca
2
þ
-sensitive signal that corresponds to the excitation of this dye at
400 nm, as advocated previously (
Ogden et al., 1995
). However, few studies to
date have explored the more commonly used dyes (Fluo-3/4 and Rhod-2) at the
longer wavelengths achievable currently with tunable Ti:Sapphire lasers (up to
1100 nm). As shown in
Fig. 8
, the Fluo-based dyes do not show the peak of
fluorescence anticipated from approximately double single-photon wavelengths
(
1000 nm). The small peaks in the excitation spectrum observed at 400-470 nm
