“Calibration-on-the-spot”: How to calibrate an EMCCD camera from its images

In order to count photons with a camera, the camera must be calibrated. Photon counting is necessary, e.g., to determine the precision of localization-based super-resolution microscopy. Here we present a protocol that calibrates an EMCCD camera from information contained in isolated, diffraction-limited spots in any image taken by the camera, thus making dedicated calibration procedures redundant by enabling calibration post festum, from images filed without calibration information.