Hello all –
I am in the process of measuring the point spread function of (1) 100 nm tetraspeck beads, and (2) Gattaquant brightness 6R beads, but am unsure about the protocol for experimentally measuring a point spread function.
What protocol do you recommend for experimental measurement of the point spread function?
Specifically – if I use a 100x/1.47 numerical aperture objective, with a sCMOS camera that has 6.5 um pixels, so that the final pixel geometry is 65 nm, then what is the recommended photon count per frame, in the z-stack?
In other words, the bead, when its in focus, shows up as a “blob” over ~3-4 by ~3-4 pixels. What would l want the ADU (analog to digital) value to be at each pixel - or, similarly, the total ADU value summed over the whole blob? Is there a standard exposure time and laser intensity (kW/cm^2)?
Have a good week,
p.s., I am also posting this in image.sc