Biology Reference
In-Depth Information
(
Shimomura, 1991
) but when it is reconstituted with apoaequorin to form an
aequorin complex (f-aequorin) the level of luminescence produced on reaction
with Ca
2
þ
is almost 20-fold higher than that produced when native coelenterazine
is used. In addition, f-coelenterazine has the highest permeability through cell
membranes (
Shimomura, 1997
).
As coelenterazine is lipophilic, apoaequorin-expressing cells, tissues, and whole
organisms can simply be incubated in coelenterazine solution. However, this
method is successful only in tissue culture cells and in simple organisms that
have a large surface area-to-volume ratio where e
usion occurs. In
more complex, multicellular organisms such as developing vertebrate embryos,
reconstituting aequorin is more of a challenge. In the case of our
a
-actin-apoae-
quorin transgenic zebrafish, we started our f-coelenterazine incubation as early as
the eight-cell stage (i.e., 1.25 hpf ) when the embryonic cells had a large surface
area-to-volume ratio, and embryos were incubated continually in this 20
m
g/ml
coelenterazine both up until, and during data collection, which took place from 16
to 48 hpf. In the case of the apoaequorin-expressing transgenic mice,
Rogers et al.
(2007)
introduced native coelenterazine into adult mice (at 4 mg/kg) by injection
into the tail vein and into new-born mice (at 2-4
m
g/g) by intraperitoneal injection.
These, and other protocols used for introducing coelenterazine into various intact
organisms and tissue culture cells are summarized in
Table III
.
Y
cient di
V
IV. Techniques for Detecting Aequorin Luminescence
erent types of equipment are commercially available that
can be used to detect or visualize aequorin-generated luminescence. These range in
capability, price, design, and commercial availability. At the lower cost end, there
is the simple test tube/culture dish luminometer, which provides only temporal
Ca
2
þ
signaling information, costs just a few hundred US dollars, and is supplied by
several di
Currently, several di
V
erent companies. At the higher cost end are several custom-designed
imaging systems, which provide both temporal and spatial luminescent informa-
tion, as well as bright-field and fluorescence images (if required), to enable the
correlation of Ca
2
þ
signaling events with morphological features and other cellular
changes. These systems are obviously a lot more costly and are built to order by a
small number of specialist companies. Some examples of the types of detectors that
have been used to image aequorin-generated luminescence are shown in
Table IV
.
It may be di
V
cult to justify the purchase of expensive single photon imaging
equipment at early stages in a project. Often a photon counting photomultiplier
tube (PMT) can be used in place of an imaging photon detector (IPD) to determine
the timing and amplitude of bioluminescence signals in living systems. By adding a
near-IR light source and an appropriate blocking filter in front of the PMT, a
relatively inexpensive near-IR sensitive camera can be used to continuously moni-
tor morphological development while the PMT reports total bioluminescence
activity. An example of this type of system is shown in
Fig. 2
A. Accurate correlation
Y
