Exciting beads for measuring the PSF

Probes & Prep
1

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,
Yossi

p.s., I am also posting this in image.sc

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Hello

Quarep-LiMi has written up a protocol for collecting point spread functions. It has been written primarily for point-scanning and spinning disk confocal systems but most of the suggestions should be applicable to you. To summarise on suggested ADU values, it’s important to have as high signal as possible without saturation to maximise signal-to-noise.

I would also encourage you to join the Quarep-LiMi WG5 working group which is concerned with collecting point spread functions if it is something you are interested in.

Best wishes
Claire

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Yes - thank you. I am also copying a screenshot of the relevant section of the protocol below.
image