*Informative Commercial Announcement*: Advancement in Quantitative EMCCD

[deepak sharma-2-3]

*Informative Commercial Announcement*: Advancement in Quantitative EMCCD

The accuracy and integrity of photometric image measurements is a point of
critical significance with regard to advanced quantitative low-light
biological imaging. Photometrics is pleased to announce a significant
advancement in the “state-of-the-art” for quantitative, on-chip electron
multiplied (EMCCD) cameras. The features incorporated into this next
generation camera, the Evolve, deliver confidence and accuracy of
quantitative measurement, increased signal to noise ratio, enhanced ease of
use and advanced functionality.

The core technology of the Evolve is a highly optimized implementation of
the established E2V 512x512 backthinned EMCCD format combined with on-board
intelligence to allow users to utilize advanced features that enhance
imaging capability as well as enable them to measure results in electrons –
all in real-time. These features are all completely under the control of the
user and offer a powerful advantage in quantitative imaging.

The Evolve also has ultimate specifications in terms of traditional camera
performance criteria and provides an ideal blend of high quantum efficiency
(>90% QE) and large well depth. The implementation is such that the basic
noise characteristics (dark noise and read noise) under conventional imaging
conditions are brought to the lowest levels presently available, thereby
expanding the dynamic range and flexibility of the platform to a wide range
of imaging conditions. Dark noise is reduced by approximately 50% for each 7
degrees drop in temperature and the Evolve is peltier cooled to -85 degrees
C  to provide a dark current specification of 0.001 e-/pixel/sec. This
benefits longer exposures under non-EM gain conditions where the Evolve has
extremely low read noise (usually lower than 6 electrons). The non-EM gain
read noise specifications have been brought to an unprecedented level of
performance on par with the most refined interline CCD cameras available.

The basic concepts of dark noise and read noise are often referred to on
camera spec sheets,  it has been ill-advised to make decisions based solely
on these basic specifications; some very important differences with regard
to image quality (and data integrity) for EMCCD cameras tend to be
undocumented specifications. An example of one of the pitfalls of not having
the entire picture would be the use of variable clock rates. By increasing
the clock-rate (rate at which data is moved off of the chip electronically
during readout) it may be possible for a manufacturer to tout a gain of a
couple frames per second at zero exposure time. The signal to noise ratio
for this increased readout rate may not be specified. What if the exposure
time has to be increased to reach an equivalent signal to noise ratio at the
sub-optimal readout rate? Is there a relevant benefit to such a feature? The
Evolve specifications do not obfuscate these important variables.  The
Evolve leverages Photometrics’ established advanced clocking enhancement
technology to enhance and optimize noise factors such as clock induced
charge and charge transfer efficiency to the lowest levels yet achieved.
This consideration provides for the highest data integrity at the fastest
speed.  

Important specifications include: clock induced charge (CIC—data corruption
resulting from imperfect clocking waveforms moving the signal through the
chip), charge transfer efficiency (CTE—causes ‘streaking’ of signal from a
pixel to adjacent pixel(s) as it is read from the chip), and field
uniformity (does the entire chip respond exactly the same to exactly the
same amount of incident signal? is there a ‘fixed-pattern’ noise
contribution?). Bias stability (stability of the offset of the signal with
zero exposure time), and gain stability (stability of amplification) are
also enormously important considerations in the context of EMCCD cameras to
be used for quantitative applications.  

Photometrics endeavors to lead the industry towards a more accurate and
reproducible approach to imaging by providing high-tolerance specifications
for sources of noise such as those mentioned above. The Evolve provides
optimized (and ground-breaking) performance for each specification. The
specifications are not derived from an exceptional ‘Golden Camera’ housed
somewhere deep in engineering. They are the rule for production cameras and
each production camera is optimized and evaluated to specification.
Responsible optimization and documentation of specifications forms the
foundation for the evolutionary next-level of quantitative functionality
incorporated into the Photometrics Evolve.

The cornerstone of any truly quantitative application involves calibration.
In the context of EMCCD cameras, the gain must be calibrated. CCD cameras
have an inherently very linear response to increasing light levels (limited
by the dynamic range) and this makes them excellent photometric devices.
Calibration ensures that a given brightness value on the image corresponds
to a reliable measurement of the ‘real-world’ value representing the number
of photons that a pixel has registered. It is of primary importance to
ensure consistent and interpretable mapping of photoelectrons to grey
levels. Without an understanding of the absolute units represented by a
brightness value, there isn’t really a quantitative measurement and the
comparison of measurements from different cameras (or at different camera
settings) becomes ambiguous at best.

For this reason, there are factors that must be considered to generate
defensible data on a quantitative EMCCD. As outlined above, the gain
(mapping of number of electrons to a brightness level) must be known. As
EMCCD chips are used and become aged, the gain amplification characteristics
change, and so it is of great importance to be able to reliably calibrate
the gain amplification at regular intervals. The Evolve incorporates an
innovative and convenient built-in light source to permit this calibration
relative to a physical standard integral to the camera without the necessity
of removing the camera from the microscope.

It is also important to linearize the gain mapping such that the
investigator knows how much amplification is being applied (e.g. 20x gain
amplifies the signal 20x, 100x gain amplifies the signal 100x). On some
previous generations of EMCCD this gain mapping was non-linear so that the
gain setting had no intuitive relation to the actual signal amplification.
The Evolve linearizes the gain mapping with easily over 5000 sample points
all the way from 1x to 1000x.

As a ground-breaking measure in quantitative EMCCD technology, the Evolve
permits real-time presentation of brightness levels in terms of ABSOLUTE
UNITS, in this case, photo-electrons. In other words, because of the
sophisticated built-in calibration and quantitative considerations, the mean
number of photo-electrons contributing to a given pixel can be presented in
real-time without arduous secondary calibrations and calculations. It should
be noted that normally a feature with a given quantum yield will appear at
different brightness levels at different gain settings even though the
number of electrons contributing to a pixel are the same. What this
technology offers is the ability to compare images taken at different gain
settings and on different cameras, in terms of absolute units. Experimental
variability is therefore brought within the limits of variability due to
noise, and the sources of noise have been carefully minimized to the current
state-of-the-art.

Spurious charge is a phenomenon that contributes to variability in pixel
values due to EM gain amplification conditions. These spurious ‘hot-pixel’
events are discrete, follow statistical probability, and can be seen as
anomalous bright pixels distributed randomly and changing position randomly
from frame to frame. Recall that point sources on a microscope are
diffraction limited, and typical intracellular image features of interest
are typically larger than the diffraction limit. What this means is that a
very bright point source arising from the sample is unlikely to contribute
only to a single pixel without having some proportional influence on the
brightness of surrounding pixels. Thus, under most circumstances, spurious
noise events can be discriminated from bright pixels that originate from the
actual image data. The Photometrics Evolve provides an on-the-fly noise
reduction algorithm that can be optionally applied to greatly attenuate
spurious charge speckling in each frame. The parameters of this filtering
operation can be adjusted interactively to reflect noise events that clearly
fall outside the limitations of the optics and into the realm of noise
artifact.  

The dynamic range is a measure of the ability to quantify very small signals
along side very intense signals without reaching the limits of the detector.
The Evolve has a vast dynamic range by virtue of the large pixel well
capacity (total number of electrons that the pixel can hold) and the very
low noise levels (determines the smallest signal that can be reliably
quantified). To adequately sample the entire dynamic range into brightness
levels, the camera must be digitized at 16-bits, or roughly 65 thousand
different brightness levels. However, the dynamic range of the sample may
differ significantly from that available to the camera, and for this reason
the Evolve incorporates an innovative ability to change the bit-depth to
better match the dynamic range of the sample. Digitizing at a lower
bit-depth means that more electrons are accumulated to transition to a
higher brightness level, this decreases the contribution of shot noise to
variation in brightness levels, makes detection of brightness transitions
and boundaries more discrete, and makes the data smaller and easier to
manipulate. An additional benefit of this variable bit depth feature in the
EMCCD world is that a user can use this feature to more accurately map ADU
units to correspond to real changes in photon levels. This can be optionally
used to make data easier to interpret by reducing the distraction of
extraneous information.


The innovations incorporated into the Evolve EMCCD were conceived with
feedback from the scientific community and are designed to meet the
requirements of demanding real-world applications. Much attention has also
been paid to ease of use, intuitive functionality, streamlined form factor
and defensible design features that provide tangible and demonstrable
benefit to the researcher. We encourage you to take a closer look and review
the online demonstrations at:

http://www.evolve-emccd.com/

Photometrics endeavors to define value in terms of driving cutting-edge
performance specifications and a responsible approach towards quantitative
imaging. Informative, candid discussion surrounding EMCCD technology is invited.


Kind Regards,

Deepak K Sharma, Ph.D

Product Manager
Photometrics