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Just a brief comment on point 1 - if your microscope is producing ghosting
artifacts (being able to 'see the grid') then the PSF you measure from the beads
is the 'correct' PSF - deconvolving with a PSF measurement which includes ghosts
*should* remove the ghosts from the real image as well, provided they relative
position of the ghosts remains constant over the field of view (probably also
subject to numeric/ noise constraints). That said, having the ghosts in the
first place is not going to make your images any better and if there' a hardware
solution I'm sure it would be worth correcting.
If you want to get a handle on the whether measured or theoretical/blind PSFs
are best in your case, you can try embedding beads in a block of agar and/or
putting beads on either side of your biological specimen.
cheers,
David
----- Original Message ----
From: Dani <[log in to unmask]>
To: [log in to unmask]
Sent: Fri, 24 June, 2011 2:22:24 AM
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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