HomeVolocityGeneralIntermediate magnification introduced by my Optivar when Calculating PSF's for data acqired using a Spinning Disk confocal system

3.36. Intermediate magnification introduced by my Optivar when Calculating PSF's for data acqired using a Spinning Disk confocal system

The Volocity software calculates PSFs in object space (sometimes referred to as the back projected plane or specimen plane or some other combination thereof). This means that they remain independent of magnification and are applied based on the physical dimensions of the images they are used to deconvolve.

This means that you don't have to enter the magnification parameter into a dialog to calculate the PSF and that PSF can handle any magnification because all of the relevant scaling is done by setting the correct calibration in the images.

To determine the PSF for a Spinning Disk system, we need to take into consideration the interference pattern due to the multiplicity of pinholes in the spinning disk unit that lies in the light path somewhere between the detector and the sample. To proceed with the calculations, we need to determine the back projected pinhole size and spacing for the spinning disk.

In determining the pinhole size and spacing for the spinning disk unit, the calculation must include all of the magnification between the spinning disk and the physical sample. This might typically only be the objective but can include an Optivar should there be one positioned between the sample and the detector when the images were acquired. Do not include intermediate magnification that does not lie between the spinning disk and the physical sample.

A physical way of checking the pinhole size and spacing is to stop the disk and measure the pattern in the image plane. The disk spacing is 250µm and the pinhole size is 50µm, so you would expect to see measurements of 2.5µm and 0.5µm for the distance and size respectively using a 100X magnification objective.

When you add an optivar you should think of it as going from an objective lens with a given focal length to a lens system composed of the objective and the optivar.

This new 'lens system' has its focal length (f) reduced by M (where M is the magnification of the Optivar) but the size of the entrance aperture into the objective remains the same. At small angles the NA is the ratio of f to the radius of the limiting (usually entrance) aperture adding that mag (Reducing f) whilst not changing the entrance aperture causes the NA (Numerical Aperture) to drop as well.