The PSF Wizard combines two steps which are necessary for extracting experimental point spread functions (PSF) from Z-Stacks of subresolution fluorescent beads:

  • A bead averaging step finds individual beads, presents them for inspection, allows you to select the ones you like and then creates an averaged combination of all selected beads. This stack shows a single bead which is, as a consequence of the averaging function, fairly free of noise.
  • The averaged bead stack is then run through the Create PSF function which removes background and residual noise, correctly scales the PSF and also converts the stack into a 32-bit floating point format which is better suitable for the mathematical procedures used in deconvolution.
  1. You have acquired a z-stack image. For more information, see Measuring the PSF Using Subresolution Beads.
  2. You have the license for the Deconvolution module.
  3. The use of the PSF wizard is activated.
  1. On the Processing tab, select the function PSF Wizard.
  2. Use Wizard and Bad Pixel Correction are activated by default.
  3. On the top of the Processing tab, click Apply.
  4. The PSF wizard opens and guides you through the creation of the PSF.
  5. If the Use wizard checkbox deactivated, the function displays the parameters for Bead Averaging. Note that these parameters are only available in Show all mode. We recommend using the PSF wizard.
  6. The result of the wizard is a PSF file which you can use for deconvolution of images acquired under the same conditions.

This method determines the position of fluorescent beads in a z-stack image. If these beads are too close to one another, they are excluded from the calculation. Beads which are far enough apart from one another are combined into a single bead, from which it is then possible to calculate a PSF using the Create PSF function.

Description of the algorithm

This function consists of a series of steps before and after processing. The aim is to find beads that are far enough apart from one another. The processing steps are as follows:

  • Select input image
  • Image smoothing
  • Segmentation
  • Alignment of the center of the found beads
  • Averaging of the beads