Biology Reference
In-Depth Information
which get amplified by an electronic circuit. The output of the circuit is monitored
by a computer for recording purposes, and the collected data is then postprocessed
to generate meaningful images (see
Fig. 3
).
Bioluminescence can be di
Y
cult to image because there is often very little signal,
Y
and because it is di
cult to predict exactly when and where the signal will be
produced. Because the signals are often quite small, it is important to use the most
e
cient optical system possible to collect the light. An overview of the main types
of optical systems used for collecting light from bioluminescent samples is given by
Karplus (2006)
and see
Fig. 4
. In microscopy, it is important to select an objective
with the highest ratio of numerical aperture to magnification (NA/mag). In macro-
imaging, it is important to select a lens with smallest possible working distance and
the lowest possible f-stop, typically known as a ''fast'' lens. It can be helpful to have
an electronic source on hand to test the e
Y
ciency of an optical system in biolumi-
nescence imaging mode and ensure that the various components of the system are
performing as expected (
Cr´ton and Ja
Y
e, 2001
).
Another important consideration for bioluminescence imaging is that the sam-
ple being imaged must be kept in a light-tight enclosure to eliminate any direct or
reflected ambient light from reaching the imaging detector, as it could easily
overwhelm the bioluminescence signal. Because the sample is normally kept in
complete darkness during bioluminescence imaging, it can be beneficial to periodi-
cally obtain bright-field and fluorescence images to determine the stage of devel-
opment or morphological condition of the sample.
Such bright-field and fluorescence images are typically orders of magnitude
brighter than bioluminescent images, so they can be obtained in a very short
period of time compared with that required to accumulate a meaningful biolumi-
nescence image. Until the recent development of deep cooled, back-thinned, elec-
tron multiplying charge-coupled devices (EMCCDs), detectors capable of single
photon imaging for bioluminescence were not well suited to acquiring bright-field
and fluorescence images because they used microchannel plates to amplify and
transmit images inside the detector. Microchannel plates blur and distort the
V
Imaging
detector and
electronics
Photon data
processing
Computer
recording
Photons
Sample
Photons
Electrons
Optics
Photon images
Fig. 3
In bioluminescent imaging, some of the photons emitted from a sample are collected by an
optical system and directed onto a detector that converts the incident photons into electrons. These
electrons are processed by electronic circuits that provide data to a computer indicating the time and
position of the detected photons. The computer program then postprocesses the photon data to generate
images.
