When comparing localization microscopy data between imaging platforms it is often convenient to express the data in terms of absolute quantities, such as photons, rather than raw units obtained from the camera. DeltaVision provides a method for accurately measuring a digital camera's conversion factor in units of photoelectrons per Analog-to-Digital Unit (e-/ADU). This measurement, along with the camera's quantum efficiency (at the wavelength used in the experiment), is used to express image intensity in terms of absolute photons instead of arbitrary intensity counts.
The measurement protocol and algorithms used are derived from European Machine Vision Association standard EMVA1288 (reference: http://en.wikipedia.org/wiki/EMVA1288).
The protocol involves collecting images at a number of discrete incident light intensity levels that are chosen to represent a range from dark current to an intensity that causes near full-well saturation of the camera's detector.
The measurement tool is available from the Resolve3D Calibration menu or Resolve3D Settings | Imaging tab. Follow the steps in the Measure Camera e-/ADU Conversion Factor window to obtain a final e-/ADU conversion. The result is displayed at the bottom of the window. This value is saved and used for further localization data analysis.
The e-/ADU conversion factor is dependent on the camera and the camera gain settings used for imaging. For instance, if the user changes their camera from a CCD camera to an EMCCD camera, it would be necessary to measure the conversion factor prior to acquiring any localization data. Additionally, if the user changes EMCCD or conventional gain settings, it would be necessary to measure the new e-/ADU conversion factor. All conversion factors are saved for each camera according to the desired camera gain setting.
Running a Localization Imaging Experiment