Camera setup

As ThunderSTORM uses real units (photons, nanometers), accurate camera parameters need to be supplied by the user. The effective pixel size can be determined from the actual pixel pitch of the camera sensor and from the total magnification of the optical setup, or by performing a spatial calibration using a stage micrometer. The detector response (photoelectrons per A/D count and base level offset of the camera digitizer) can be obtained from the specification sheet supplied by the camera manufacturer or from photon transfer curve measurements (the dependence of the variance on the mean value of pixel intensities), see e.g., [1, 2]. Correct pixel size is important for proper spatial calibration of the rendered images. Camera conversion gain and offset influence simulated images and the estimates of localization precision.

Pixel size

Pixel size is used to convert molecular coordinates from pixels to nm. It is the size of one camera pixel as projected to the sample plane.

Conversion of image intensity to photoelectrons

For CCD cameras, the image intensity is converted to the number of photoelectrons according to the formula

i=p\left(I-b\right)\,,

and for EMCCD cameras, the conversion is according to the formula

i=\frac{p}{g}\left(I-b\right)\,.

Here i is the number of photolectrons, p is the CCD sensitivity (in photons per A/D count), g is the EM gain for EMCCD cameras, I is the image intensity (in A/D counts), and b is the base level specifying the camera digitizer offset (in A/D counts). If the EM gain checkbox is not selected, a CCD camera is assumed.

Note that the outcome of setting the EM gain to 1 and unchecking the EM gain checkbox is different as the localization uncertainty of molecular positions is computed using different formulas for CCD and EMCCD cameras. Also a stochastic nature of EM gain is modeled in the generator of simulated data.

References

  • [1] J. Janesick, K. Klaasen and T. Elliott(1985) CCD charge collection efficiency and the photon transfer technique, pp. 7–19. Cited by: Camera setup.
  • [2] L. J. Van Vliet, F. R. Boddeke, D. Sudar and I. T. Young(1998) Image detectors for digital image microscopy, Digital Image Analysis of Microbes: Imaging, Morphometry, Fluorometry, and Motility Techniques and Applications, pp. 37–63. Cited by: Camera setup.