Biology Reference
In-Depth Information
images they transmit, have a limited dynamic range, and can also be permanently
damaged if they are exposed to too much signal. While a nonintensified detector
could be used to acquire bright-field and fluorescence images, the problem of blur
and distortion in the bioluminescence image would still remain. In addition, it was
a challenge to adjust the size and registration of images acquired from two di
V
erent
detectors.
An EMCCD sensor can be fabricated on a substrate alongside one or more
amplifiers with programmable gain settings, so a computer can rapidly change
from a low-gain to a high-gain setting. Thus, a computer-controlled EMCCD is
capable of acquiring bright-field and fluorescence images at low-gain settings, as
well as bioluminescence images at high-gain settings (see
Fig. 4
). This makes it
possible to eliminate blurring and distortion in all three types of images to be
acquired, improving the spatial resolution in the bioluminescence images by 2-3
times over what can be achieved with a microchannel plate based detector. Because
all three types of images can be obtained with the same sensor, the scale and
registration are identical as well. Furthermore, back-thinned EMCCDs have a
significantly higher quantum e
ciency in the visible light spectrum, compared with
photocathode materials used in intensifier-based detectors, so they are able to
respond more e
Y
ciently to weak signals.
While EMCCDs are capable of detecting single photon events when the electron
multiplying gain is high enough to overcome the read noise of the output amplifier,
the electron multiplying gain mechanism is subject to substantial statistical varia-
tion. For example, when an output signal of 1000 electrons occurs, it is easily
detected as a meaningful event, but it is not possible to be certain how many input
electrons generated this signal—it may have been just 1, or 2, or 5 input electrons.
As a result, EMCCDs operating in photon counting mode have limited ability to
track large intensity changes. The maximum signal intensity that can be recorded
reliably is essentially determined by the frame rate at which the sensor is read out.
The range can be extended at the expense of the field of view by selecting a small
region of interest, and/or at the expense of spatial resolution by binning together
adjacent pixels on the sensor.
Two additional limitations of EMCCDs arise from the circuitry used to read out
the image data. First, in order to record the signal detected by the CCD sensor,
Y
systems, respectively. The EMCCD can be modified to acquire bioluminescence information as well as
bright-field and fluorescence images. On the other hand, a resistive-anode Imaging Photon Detector
(RA-IPD) can be used in conjunction with a CCD camera, the latter to acquire bright-field and
fluorescent images, when a higher dynamic range and temporal resolution are needed for the biolumi-
nescence signal. For both the EMCCD and RA-IPD-based imaging systems, a high level of automation
for the microscope makes it possible to rapidly switch between the various imaging modes, and also
makes it possible to have the computer run automated acquisition sequences over extended periods,
typically overnight. The motorized focus allows the computer to acquire image stacks in any imaging
mode for three dimensional reconstructions. Both systems were designed and built by Science Wares
Inc., Falmouth, MA, USA.
