Technical Note

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 whilst minimizing quantization error, the camera is 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 our online demonstrations, tools/calculators, and technical notes. Photometrics endeavors to define value in terms of driving cutting-edge performance specifications and a responsible approach towards quantitative imaging.

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