Biology Reference
In-Depth Information
60 s
Mito
Cyto
Caf
0-Ca
Fig. 17
Simultaneous measurement of Ca
2
þ
signals in the cytosol and in mitochondria. A rabbit vagal
sensory neuron was incubated with 1
m
M each Rhod-2 AM and Fluo-3 AM for 1 h at 23
C and then
superfused with nominally Ca
2
þ
-free physiological saline (0-Ca). Rhod-2 and Fluo-3 fluorescence,
excited at 543 and 488 nm, respectively, were imaged simultaneously by laser-scanning confocal micros-
copy. Data are represented as fluorescence change relative to baseline (
D
F/F
0
). A 5-s pulse of ca
eine
(Caf, 10 mM) was delivered by superfusion. The durations of reagent applications are indicated by the
bars at the bottom.
V
C. Monitoring Cytosolic and Mitochondrial [Ca
2
þ
] Simultaneously
Because indicators whose AM esters are uncharged load primarily into the
cytosol, while Rhod-2 preferentially loads into mitochondria, one can monitor
Ca
2
þ
signals in the two compartments simultaneously. For measuring cytosolic
[Ca
2
þ
], one should select an indicator whose excitation and emission wavelengths
do not interfere with Rhod-2 measurement. Since Rhod-2 is a rhodamine-based
indicator, a fluorescein-based indicator would be suitable for the cytosolic mea-
surement (e.g., members of the Fluo family of indicators).
Figure 17
shows an
experiment where the cytosolic and mitochondrial Ca
2
þ
signals are monitored
simultaneously in a vagal sensory neuron being stimulated with a brief pulse of
ca
eine (an agonist that activates ryanodine receptor Ca
2
þ
channels to release
Ca
2
þ
from Ca
2
þ
stores in the ER). The cytosolic and mitochondrial Ca
2
þ
transients have very di
V
erent decay kinetics: the time for the Ca
2
þ
signal to
V
decay by 80% was t
80%
¼
7.7 s in the cytosol and t
80%
¼
65.1 s in mitochondria.
VIII. Concluding Remarks
Fluorescent Ca
2
þ
indicators have contributed enormously to our understanding
of intracellular calcium regulation. For those who are beginning to use these
indicators, the technical details can seem bewildering. This compendium of
