3. EMCCD vs ICCD – experimental comparison
The EMCCD
Andor Technology iXon DV887 back-illuminated (up to > 90% QE). 512x512 frame-transfer
sensor, 16 µm2 pixel size, capable of delivering up to 34 full
frames/sec.
The ICCD
Stanford Photonics XR Mega 10 ICCD, containing a GenIII+ intensifier and 1280 x
1024 CCD sensor, 6.7 µm2 sensor pixel size, capable of 15 frames/sec
at full frame readout. The sensor is fiber-coupled to the intensifier tube, with
a ~ 1.5 to 1 tapered fiber, yielding an effective pixel size of >10 µm2.
Binning (2x2) of the ICCD was used to enable shorter exposure times (faster frame
rates) to be employed in this camera, whilst also making the effective pixel size
more comparable to that of the EMCCD (better for more accurate sensitivity comparisons).
Such binning should result in more favourable S/N performance (a 2x2 binned superpixel
collects 4x more light that a non-binned pixel).
The comparison
In order to perform a direct performance comparison between the BV-EMCCD camera
and the Gen III+ ICCD, a series of Fluo-4 loaded rabbit urethra smooth muscle cells
were imaged using a Nipkow confocal spinning disk, taking a short kinetic series
(10 frames) with one camera, then performing the same analysis with the other camera
directly after with the same cell still in place. The order of which the images
were recorded was alternated for each subsequent cell, in order to rule out photobleaching
effects on the differing S/N levels. In general, largely due to the rapid confocal
nature of the technique and the short 33 ms exposure times employed (2x2 binning
of the ICCD was necessary in order to achieve this exposure), the fluorescent images
represent extremely low photon fluxes. All cells imaged were within the same field
of cells and subjected to identical dye loading times. However, it was obvious that
some cells were inherently weaker than others. This is due to slightly different
loading efficiencies, focal fine position and also, the laser power was purposefully
varied slightly in order to produce different degrees of signal intensities. The
important point was that each cell was imaged ‘like-for-like’, varying only the
camera.
(A) Comparative raw and smoothed low-light fluorescent images recorded of a live
smooth muscle cell using the BV-EMCCD and the Gen III+ ICCD
(B) Further comparative cell images of weakly loaded cell, representative of an
image close to the ‘photon shot noise’ detection limit; 33 ms exposure time per
frame; high EMCCD or ICCD gain setting throughout to eliminate read noise.
Figure 1(A) shows one of the cells recorded with each camera type, both before and
after applying a smoothing algorithm. It is clear that both signal contrast and
resolution is markedly better in the case of the images recorded with the BV-EMCCD.
The effect of smoothing is to enhance the contrast further between fluorescent signal
and background, at the expense of some resolution. However, even after smoothing
of each image, the same improvements are noticeable in the BV-EMCCD images compared
to the corresponding ICCD image. Figure 1(B) shows a further representative cell
comparison from this experiment, this one of a weakly loaded cell representing a
signal intensity that is close to the ‘photon shot noise’ detection limit for this
exposure time. It is clear that signal intensity and image contrast is markedly
better for the image recorded with the EMCCD, a direct function of a higher S/N
ratio afforded by the higher QE of the EMCCD detector (both types of detector have
overcome the read noise detection limit). The photon flux was so low that this cell
could barely be detected at all with the ICCD, but is certainly identifiable with
the EMCCD. The superior signal of the EMCCD is to be expected given the markedly
restricted QE of ICCDs compared to the unrestrained QE of the BV-EMCCD. Indeed,
at the 516nm max. of Fluo-4 dye, the BV-CCD should have > x2 QE than a GenIII+
photocathode. This should result in > x1.4 increase in Signal-to-Shot Noise ratio.
Furthermore, the resolution from the BV-EMCCD is markedly better, and one can for
example more easily identify areas of intracellular dye compartmentalization. This
resolution improvement arises to a large extent from the different electron amplification
technology used in the EMCCD, which virtually eliminates the pixel cross-talk that
is inherent in ICCD measurements (yielding a form of instrumental blurring), but
also from the slightly larger area of the 2x2 binned pixel required to run at a
faster readout speed (and shorter exposure time). In comparison, the EMCCD produces
images of stark resolution – post-acquisition smoothing can be applied artificially
through software if required but this is the researcher’s choice. It is interesting
also to note that, in terms of sensitivity, a greater number of photons should hit
the slightly larger pixel area of the 2x2 binned ICCD than that of the non-binned
BV-EMCCD, yet the latter camera clearly maintained a clear S/N advantage (without
the need to sacrifice resolution for speed).
It is important to note that all of the images shown here are of extremely weak
fluorescence confocal signals, and would be lost entirely within the read noise
floor if it were not for the respective signal amplification technologies.
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4 Other detector technologies?