James Joubert, applications scientist at Photometrics, provides advice on how to deal with variability in camera response.
Quantitative comparison of image data between experiments on different days or at different labs is challenging.
One reason is the variability in camera response depending on settings and camera.
Scientific cameras commonly report data as arbitrary grey levels, which should be linearly proportional to the amount of detected light.
However, the proportionality between grey levels and detected light varies among manufacturers and even among the same model cameras.
Quantitative comparison requires removal of this variability, which can be achieved by using a standard unit in place of the grey level.
Since all CCDs generate photoelectrons when light hits the sensor, the photoelectron is the optimal candidate.
Furthermore, by simply multiplying a typical grey level image by a camera’s specific proportionality constant (known as gain), the image can be converted to photoelectrons.
This conversion factor can be determined easily by measuring the mean and variance of a flat field image for any CCD camera.
This is described in more detail in the Mean Variance whitepaper which can be downloaded above.
EMCCDs require another step due to additional EM gain. For a properly calibrated EMCCD camera, this conversion factor can be obtained for any EM gain by first measuring the factor at zero EM gain and then dividing by the exact EM gain used.
For example, the Evolve camera from Photometrics provides such conversion in real-time with its Quant-View feature.
One example of the usefulness of a standardised unit is normalising for variation in fluorescent protein transfection levels.
Standard photoelectron measurements enable researchers to discern whether cells are being transfected to similar levels, day-to-day and even year-to-year.
Overexpression artifacts can be identified for improved sample selection, and as stated earlier, a standard photoelectron unit makes data comparison easier even between labs.