Biology Reference
In-Depth Information
there must be a period of dead time while the accumulated image data is shifted out
of the active area of the sensor. Second, the signals used to shift the image data
from the active area into the readout amplifier can also impose noise on the output
that looks identical to single photon events in the photon counting mode. Signifi-
cant advances have been made to minimize the noise (called clock-induced charge)
that is generated. However, these two e
ects are still significant such that increas-
ing the readout rate beyond a certain level actually degrades the net single photon
imaging performance of the EMCCD.
In situations where significant bioluminescence intensity changes are taking
place over periods less than a second, an intensifier-based detector is likely to be
a better choice than an EMCCD (see
Fig. 4
). The spatial resolution of commer-
cially available intensifier-based detectors is usually in the order of tens of microns
at the detector input window, which is adequate for many applications, but not as
good as what can be achieved with commercially available EMCCD detectors,
which typically have a pixel size of 8 or 16
m
m. There are two main types of
intensifier-based detectors, those with a phosphor image output that is optically
coupled to a visible light CCD, and those with an electrically encoded anode that
produces position sensitive pulses for each detected event. The temporal resolution
of detectors with an optically coupled phosphor output is typically in the range of
tens of milliseconds due to the persistence time of the phosphor and the frame rate
of the CCD.
The best temporal resolution for single photon imaging can be in the order of
tens of nanoseconds, and is achieved only by intensifier-based detectors with an
electrically encoded anode. The dynamic range of such detectors is constrained by
the dead time of the pulse processing electronics, which is typically in the order of a
few microseconds per detected event. Improvements continue to be made in the
spatial resolution of microchannel plates and throughput speed of encoded anode
detectors (
Lapington, 2004; Siegmund et al., 2005
), but the cost and complexity of
operating such detectors has prevented them from being used widely for biolumi-
nescence imaging so far.
Another factor that should be considered when selecting a detector for biolumi-
nescence imaging is the expected signal-to-noise ratio of the recorded image data
(
Karplus, 2006
). Frequently, accepting lower spatial resolution can result in a
better signal-to-noise ratio. In situations where a fast or brief signal needs to be
identified with high temporal accuracy, a photocathode detector is often capable of
a better signal-to-noise ratio than an EMCCD. Even though an EMCCD detector
can have 2
V
ciency than a detector with a photocathode,
at the high readout rates needed for good temporal resolution, incident photons
can still be lost during the dead time needed to transfer image data into the readout
frame of the EMCCD, and the photons that are detected can be obscured by clock-
induced readout noise.
Figure 5
shows a comparison of the bioluminescence
images acquired by an EMCCD and an RA-IPD photon imaging system at two
di
-20
higher quantum e
Y
V
erent stages of zebrafish development.
