Unravelling Sensitivity, Signal to Noise and Dynamic Range – EMCCD vs CCD

4. Effects Of Background Photons On Detection Limit

4.1 Background Photon Considerations

  • Deep-cooled, high-end EMCCDs represent the most sensitive detectors available on the market and perform exceptionally well in ultra low-light experiments. It is crucially important to understand however that under such conditions, it is not enough simply to have the most sensitive detector, but it is essential also to restrict as far as possible the amount of undesirable background photons that are reaching the detector.
  • Some sources of background photons may be
    • Excitation light
    • Scatter from microscope slides
    • Unblocked ambient light
    • Light from outside of the z focal plane of the imaged specimen
    • Unspecific binding of fluorescently labelled biomolecules.
  • Background photons, if not minimized, have the potential to contribute significantly to the noise floor and yield a detection limit that is defined by the background shot noise, rather than the read noise and dark current sources of the detector. Depending on the flux of background photons and also the technology used (standard CCD or EMCCD), this contribution can affect the detection limit with varying severity.
  • It is important to learn to recognise the degree of contribution of background photons to the detection limit. This is accomplished first by defining the true camera noise floor in total light tight conditions, using the same exposure times/gain levels that are to be used in the actual measurement. True light tight conditions can be achieved by screwing a blanking plate onto the c-mount adaptor of the camera. The ‘2D Line Intensity Profile’ within the Andor MCD software is very useful for reading the instrument noise characteristics.

4.2 CCD Cameras and Background Photons

Following the procedure given above for defining the true instrument noise floor, the intensity profile at any point across the image should represent largely read noise with a small contribution from darkcurrent noise (higher contribution at very long exposures). The read noise amplitude in counts can in fact be predicted from the pre-amplifier gain, available on the specification sheets that accompany Andor cameras.

Upon exposure to the signal, it only requires background photoelectron levels of say > 5 to 20 electrons/pixel for the noise floor to begin to become dominated by photon shot noise characteristics. If this is the case, both the detection sensitivity and the dynamic range become compromised.

An increase in QE is unlikely to help significantly in this case since it will make the camera more sensitive to background photons also (unless the background photons are of a different wavelength to the signal photons).

4.3 EMCCD Cameras and Background Photons

The EMCCD cameras, representing the world’s most sensitive CCDs, are particularly susceptible to background photon noise when EMCCD gain is turned on. Never forget that the EMCCD is capable of detecting even a single photon!

There are a couple of different levels of background photon noise to watch for under high EMCCD gain:

1. Background photons eliminated

this is rarely the case in an experimental set-up but it can be simulated with a good blanking plate or shutter over the sensor and keeping exposure times to an absolute minimum. The noise profile in this case should consist of a read noise base level, approx 10 counts peak to peak with the iXonEM+ range, with occasional spurious CIC noise spikes amplified well above this level (darkcurrent spikes are negligible). If the EM gain were to be turned down, the CIC spikes should disappear leaving only the read noise floor, of the same peak to peak magnitude as with gain applied.

2. Some background photons

many Total Internal Reflection Microscopy set-ups or confocal microscopy set-ups will create this limited background situation, i.e. the illumination plane in each configuration is highly restricted, drastically reducing the acceptance of background photons from outside the focal plane. Nevertheless, background photons will still be present to varying degrees and can be recognised as additional ‘spurious’ spikes alongside the expected level of CIC spikes. It is still essential to eliminate CIC and darkcurrent contribution as far as possible.

3. Background shot noise dominated

If insufficient measures have been taken to reduce background levels, it is likely that the noise profile, under conditions pf applied EM gain, will be dominated entirely by background shot noise. Under highest EM gain setting, this will have a peak to peak value of several hundreds or even thousands of counts and this magnitude will decrease as EM gain is reduced, possibly to a point where it is buried in the read noise when gain is low or off.

This is a somewhat less desirable situation for optimal operation of EMCCD technology – the EM gain may have been used to amplify a weak signal clear of the read noise, but the background photon shot noise will be amplified also and application of further EM gain will do nothing to improve this limiting Signal/Background Noise ratio.

That is not to say that EMCCD are not still highly effective in optimizing most low light signals, whether limited ultimately by background photons or not. Even set-ups such as widefield fluorescence microscopy (where the limiting background is from out of plane fluorescence) can still benefit enormously from application of EM gain, as the read noise floor can still contribute strongly (often overwhelmingly) in its absence.