Biology Reference
In-Depth Information
of bioluminescence signals with morphological events facilitates planning, optimi-
zation, and standardization of protocols for subsequent imaging experiments.
Over the past few years, several novel aequorin-based detection system designs
have been reported. One is the two-channel luminometer, which was made for
dual-wavelength aequorin measurements (see
Fig. 2
B). This system was designed
by
Manjarr´s et al., 2008
and built by Cairn Research (Faversham, UK). Here, the
luminescence emission from two spectrally distinct aequorins (GFP-aequorin and
RFP-aequorin) that are coexpressed in di
erent subcellular locations within the
same cells is divided by a dichroic mirror and the resulting beams of light are
filtered at 535 and 630 nm and then collected by two separate PMTs (
Manjarr´s
et al., 2008
).
Another new Ca
2
þ
detection device is an imaging system capable of acquiring
real-time bioluminescence data from living (and unrestrained) small animals such
as mice (see
Fig. 2
C). This system was designed by
Roncali et al. (2008)
. It is based
around a Photon Imager
TM
intensified CCD camera (Biospace Lab., France)
operating in a photon counting mode. The ICCD camera is set on top of a light-
tight chamber and records optical signals at a video rate of 25 Hz. Motion can also
be monitored by using two cameras, one that records the signal of interest and the
other that is used to video-track the animal. The latter can be achieved by
illuminating the field of view with infrared light. The signals from both cameras
are recorded simultaneously and electronically synchronized. A detailed descrip-
tion of this equipment is given by
Roncali et al. (2008)
. A similar approach has
been used with microscope-based imaging systems to continuously acquire biolu-
minescence image data emitted at short wavelengths while using longer wavelength
illumination to simultaneously record transmitted light images that show mor-
phology (
Speksnijder et al., 1990
).
One of the most recent developments in bioluminescence detection involves
using an EMCCD detector for single photon imaging (
Martin et al., 2007;
Rogers et al., 2008
). The best bioluminescence imaging detectors are capable of
single photon detection, and this requires that the detector somehow amplify
the detected signal above background noise. All electronic imaging detectors
ultimately convert incident photons from the sample into detected electrons,
V
information from cells expressing GFP- and RFP-aequorin in di
erent organelles. The system was
designed by I. M. Manjarr´s, P. Chamero, M. T. Alonso, and J. Garc´a-Sancho (Universidad de
Valladolid and Consejo Superior de Investigaciones Cient´ficas, Valladolid, Spain), and B. Domingo,
F. Molina and J. Llopis (Universidad de Castilla-La Mancha, Albacete, Spain), and was built by Cairne
Research (Faversham, UK). (C) A photon counting-based system, with a video monitoring function
(via an IR-sensitive camera), for whole-body optical imaging of un-restrained, freely moving small
animals, such as mice. The system was designed by E. Roncali, K. L. Rogers and B. Tavitian (Labor-
atiore d'Imagerie Mol´culaire Exp´rimentale, INSERM U803, Orsay, France) and M. Savinaud,
O. Levrey and S. Maitrejean (Biospace Lab, Paris). Panels (B) and (C) are modified from
Fig. 1
in
Manjarr´s et al. (2008) and Roncali et al. (2008)
, respectively.
V
