Biology Reference
In-Depth Information
ester is used to load cells, Rhod-2 loads well into mitochondria; up to 80% of the
intracellular dye is located in these organelles. The use of Rhod-2 to monitor Ca
2
þ
signals in mitochondria is outlined in
Section VII
.
A choice of ratiometric indicator can be made on practical grounds. Typically,
Fura-2 is excited alternately at two di
V
erent wavelengths, whereas the emission is
collected at a single fixed wavelength. Therefore, the pair of intensity measure-
ments, whether in imaging or in single-cell microfluorometry, must be collected
sequentially. Indo-1, on the other hand, usually is excited at a fixed wavelength
whereas emission is monitored simultaneously at two di
erent wavelengths, that is,
emission from the Ca
2
þ
-bound and Ca
2
þ
-free forms of the indicator can be
collected simultaneously. Therefore, Indo-1 potentially can give better temporal
resolution. However, in conventional imaging, Indo-1 can be more di
V
cult to use
because the two emission images, usually collected through slightly di
Y
V
erent
optical paths, can be di
cult to keep in spatial registration. Fura-2 has been the
most widely used ratiometric Ca
2
þ
indicator, both in conventional imaging and in
single-cell measurements. Indo-1 has, however, been used successfully in UV laser-
scanning confocal imaging applications (
Motoyama et al., 1999; Niggli et al., 1994;
Sako et al., 1997
). Fura Red
TM
is touted as a ratiometric indicator whose excita-
tion and emission wavelengths are both in the visible range. This indicator su
Y
V
ers
0.013 in the Ca
2
þ
-free
from having very low fluorescence quantum e
Y
ciency (
form; J.P.Y. Kao, unpublished results
4
). Fura Red di
ers from the other ratio-
metric indicators because its fluorescence intensity decreases upon binding Ca
2
þ
.
The relatively low quantum e
V
ciency implies that higher indicator concentrations
and/or higher excitation light intensities are required.
The dextran-conjugated dyes are biopolymers with pendant indicator molecules.
The dextran-conjugated indicators listed in
Table I
are available with dextran
molecular weights of 3000, 10,000, or 70,000 (Invitrogen Corporation, Molecular
Probes Brand). Being membrane-impermeant, dextran conjugates must be loaded
into cells by an invasive technique such as microinjection. Whereas the monomeric
indicators can leak out of cells at a steady rate (
Section III.C
), dextran-conjugated
indicators tend to have long residence times in cells. Therefore, dextran-conjugated
dyes can be useful in applications in which long-term monitoring of [Ca
2
þ
]
i
is
required. Instances also occur in which cells rapidly transport monomeric dyes
into internal organelles (
Hepler and Callaham, 1987
) but do not do so when dextran
conjugates are used. Because the conjugates are made by covalent attachment of
monomeric indicators to dextran polymers, individual indicator monomers can
reside in slightly di
Y
erent local microenvironments on the polymer. Therefore, the
conjugates, rather than having a unique K
d
and identical spectral properties, are
characterized by a range of microscopic K
d
s and a distribution of spectral proper-
ties. These characteristics provide a likely explanation for lot-to-lot variations in K
d
and spectral characteristics.
V
ciency of the Ca
2
þ
-free form of Fura Red was determined relative to carboxy-
4
The quantum e
Y
SNARF-1.
