Biomedical Engineering Reference
In-Depth Information
Most synthetic organic probes for ratiometric imaging are excited with UV light,
which can be damaging to cells and requires a specialized laser for confocal imag-
ing systems. Unfortunately, there are no available ratiometric Ca
2+
sensors that have
absorption within the visible range. However, it is possible to achieve effective rati-
ometric measurements in the visible-light range using two singlemetric probes like
fl uo-3 (or fl uo-4) and Fura Red (Lipp and Niggli
1993
; Speier et al.
2008
) in the
same cell(s). Both Fura Red and fl uo-3 can be excited at the same visible wave-
length (488 nm) but have different [Ca
2+
]
i
dependencies. Therefore, if both dyes are
simultaneously loaded into target cells, and two fl uorescence images are recorded at
530 nm (fl uo-3) and at 680 nm (Fura Red) using a spectral splitting system, then a
change in [Ca
2+
]
i
is indicated as the ratio of these fl uorescence intensities (
F
530
/
F
680
).
However, because the fl uorescence of Fura Red is much weaker than that of the
other visible-light excitable Ca
2+
dyes, it is necessary to use a much higher concen-
tration of the Fura Red than of the paired indicator.
In summary, in order to take full advantage of the potential benefi ts of Ca
2+
indi-
cators in studies of neural activity, investigators must pay careful attention to their
requirements for temporal resolution and sensitivity, select an appropriate indicator,
and then consider the design of the experimental protocols, sample preparation, and
confi guration of the optical recording system.
5.3
Experimental Protocols
5.3.1
Dye Loading
The synthetic organic Ca
2+
probes are negatively charged and impermeable to cell
membranes. There are two general methods for loading Ca
2+
indicators into nerve
cells: microinjection to a single cell and bulk loading to large populations of cells
(Fig.
5.2
). For loading into single cell, a grass micropipette or patch electrode fi lled
with the Ca
2+
dye of potassium salt is inserted into a target cell or applied for whole-
cell recording, and the Ca
2+
indicator is diffused directly into cytoplasm (Fig.
5.2a
).
When using a sharp microelectrode, the Ca
2+
dye can be loaded electrophoretically
with depolarizing current injection. The microinjection method for staining a single
cell can provide a very bright image of the cell over the background, achieving a
high signal-to-noise ratio for the optical recordings. This method is preferred for
measurements requiring high spatial resolution, such as recording the Ca
2+
tran-
sients in subcellular regions like dendritic branches or dendritic spines. Further, this
method enables simultaneous measurement of [Ca
2+
]
i
changes with electrophysio-
logical recording of action potentials or synaptic currents. This approach has yielded
signifi cant fi ndings related to synaptic integration in dendritic arborization.
There are several techniques for the bulk loading of the Ca
2+
dye into larger
populations of nerve cells. The most popular method for loading fl uorescent indica-
tors is by the application to the tissue of acetoxymethyl (AM) ester, which is a
