Stability in EMCCDs
EMCCDs are susceptible to various sources of data instability. Each of these sources
have been addressed in iXon Ultra and iXon3 to ensure reliable quantitative performance
throughout a kinetic acquisition and also repeatability between measurements.
The baseline (or bias level) is an electronic offset added to the
output signal from the EMCCD sensor to ensure that the displayed
signal level is always a positive number of counts. No actual noise
is associated with this positive counts value and thus it is important
to recognise that it does not affect sensitivity. However one must
remember to subtract the baseline offset value from the signal
intensity when performing signal to noise calculations.
Traditionally, when acquiring data, small changes in heat generation
of the driving electronics within the detector head may cause some
drift in the baseline level. This is often particularly observable during
long kinetic series.
Since 2002, Andor have been addressing this undesirable effect in our
high-end EMCCD cameras.
Any drift in the baseline level can be corrected by using our innovative
Baseline Clamp option. Baseline Clamp corrects each individual
image for any baseline drift by subtracting an average bias signal
from each image pixel and then adding a fixed value to ensure that
the displayed signal level is always a positive number of counts. As
such, the baseline remains at a rock-steady value during a fast kinetic
series, as shown in Figure 1.
Note: The baseline bias level is also susceptible to variation at
different EM gain settings. Again the iXon baseline clamp corrects
for this, ensuring the bias level is clamped no matter what EM gain
setting is selected.
Electron Multiplication (EM) and Temperature Stability
It is a well recognized fact that EM multiplication factor is temperature
dependent. That is why iXon pioneering RealGainTM – a linear and
quantitative EM gain calibration - is temperature compensated, i.e.
the same precise correlation between software EM gain selection and
actual EM gain holds at whatever cooling temperature is selected.
This will be addressed in more detail later.
However, another by-product of this temperature dependence is that
we must also pay close attention to optimized temperature stability
regulation. The iXon Ultra maintains a thermostatic precision of +/-
The results of such attention to detail are best understood through
observation of the stability via measurement of a stable light source,
as shown in Figure 2.
Here we used the back-illuminated iXon Ultra 897 to measure signal
from a stable LED source overlayed with a resolution chart, imaged
in conditions of zero ambient background light (use of a light-tight
Kinetic series were recorded over 500 frames @ 55 fps (17.8 ms
exposure time), frame transfer mode, Baseline Clamp activated (such
that absolute bias stability is in place also). A moderately intense
signal, such that instability would not be lost in the signal shot noise,
was recorded with x300 EM Gain.
A Region of Interest kinetic plot was derived from the data, as
shown in Figure 2. Significantly, there is no additional relative signal
variation observable over the duration of the kinetic series.
Be careful of light source instability
When performing stability measurements, care must be taken to
assess the stability of the light source. As an example, an iXon
897 was mounted on a research grade widefield epifluorescence
microscope and fixed cells, immuno-stained with fluorescence dyes
(Invitrogen Molecular Probes), were imaged over a similar kinetic
series, as shown in Figure 3.
In these experiments the variation of the signal intensity was
significant, whether EM gain was on or off; the amplitude of variation
was much higher than the shot noise on the signal. This indicates
that the light source of the microscope itself can often be subject
to much greater stability variations than could be derived from any
EM gain instability observations. One needs to very carefully check
for all sources of undesirable signal fluctuation, such as stability
of illumination (background illumination fluctuations can also
contribute) when conducting quantitative time course experiments.