Commercial Response from Media Cybernetics:
I'm a software developer on the Autoquant product.
2. The issue is not strictly theoretical vs. calculated but more of a question of how well you can measure or calculate the PSF. For measured the issue is getting a picture of a bead under the same conditions (depth and RI) as the sample. For theoretical it is knowing the values of the depth and sample RI. Can you get a picture of a bead under conditions similar to the real experiment?? Or alternatively do you know a good estimate of depth and sample RI?? Both can be tricky.
And actually MLE can be used with theoretical PSFs. You may be thinking of the adaptive PSF MLE in Autoquant. It's a version of MLE in which the PSF is allowed to change (subject to constraints). Adaptive MLE uses a theoretical or measured PSF as a starting point. Autoquant also has routines to detect spherical aberration (which can be problematic with thick samples).
Please feel free to contact us off list if you have any further questions.
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Dani
Sent: Thursday, June 23, 2011 10:22 AM
To: [log in to unmask]
Subject: Deconvolving Spinning Disk Images
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Hi there,
I would like to deconvolve my spinning disk images for quantification
purposes. Regarding this, I have a few issues/questions.
1. I have been trying to image fluorescent beads, in order to obtain PSF
measurements for my microscope (conditions). However, whenever I do this, I
always “see the grid” of the spinning disk unit, meaning that there are
multiple points visible at the same time, even when a single bead is imaged.
This only becomes obvious after severe rescaling and the other grid dots
have a very weak intensity compared to the main dot (at least 2 orders of
magnitude). I have tried synchronizing imaging time with spinning disk
speed, long/short exposures, high/low laser intensities, with/without EM
gain, etc. I always see the same thing.
The questions are: Will this affect the deconvolution efficiency/accuracy?
Does anyone have any solution for this problem?
2. I have read conflicting opinions on performing deconvolution using
experimental vs. theoretical PSFs vs maximum likelihood estimations (MLE).
Some say that when imaging thick samples (in my case ~30um) it is better to
do MLEs, because the PSF changes depending on the depth that you image, and
measured PSFs are always on the surface of the slide. Others say that
measured PSFs will always (usually) be better as they don’t assume anything
but actually measure what is going on. Then again, I have been considering
doing theoretical PSFs, as I have been having a lot of trouble actually
measuring a proper PSF in my microscope (see point 1).
Does anyone have any experience with this or can advise me on these issues.
Which programs/algorithms/plugins could I best use for this?
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