*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Commercial response from Huygens-SVI

Dear All,

Your 1st question:
The circular pattern is probably caused by bad synchronisation for which 
you already did a number of smart tests. Normally such a pattern is 
difficult to see in bead images so it looks like a real problem. I'll 
email you off-line so that you can upload the data-set so that we can 
help find the reason.

Your 2nd question:

I agree with Brian.  In the Huygens software, the MLE can be used both 
with the measured and the (on the fly) theoretical PSF so the MLE 
algorithm is actually not a deciding factor.
The deciding factors are indeed mainly the thickness and also the 
possibly different Refractive Indices for lens and medium.

The rule of thumb is that up to 10 micron thickness  the measured PSF 
can still be used. With thicker samples usage of the (on the fly) 
theoretical PSF is usually better.  Still with identical RI's with 
embedding media like Entallan or Aquatex you may go further than 10 micron.

So generally speaking a measured PSF is better than a theoretical PSF. 
And as both spinning discs and confocal can suffer from bad alignments 
that's an even stronger argument in favour of the measured one. In 
Huygens you could apply for the first 10 micron the measured PSF and for 
the remainder of your image the theoretical.

With best wishes,


Gitta Hamel

****************************************
Gitta Hamel
Managing Director Scientific Volume Imaging bv
Developers of the *HUYGENS* software
The Netherlands
phone: ++ 31 35 6 42 16 26
*****************************************


^SVI Customer support: mail us your questions [log in to unmask] 
<mailto:[log in to unmask]>or find answers online in our Huygens 
WIKI:www.svi.nl/FrontPage <http://%20www.svi.nl/FrontPage>

On 06/23/2011 05:18 PM, Northan, Brian wrote:
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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?
>
>
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Deconvolving-Spinning-Disk-Images-tp6508446p6508446.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
> ######################################################################################
> CONFIDENTIALITY NOTICE:
> This email transmission and its attachments contain confidential and proprietary information
> of Princeton Instruments, Acton Research, Media Cybernetics and their affiliates and is
> intended for the exclusive and confidential use of the intended recipient. Any use, dissemination,
> printing, or copying of this transmission and its attachment(s) is strictly prohibited. If you
> are not the intended recipient, please do not read, print, copy, distribute or take action in
> reliance upon this message.  If you have received this in error, please notify the sender immediately
> by telephone or return email and promptly delete all copies of the original transmission and its
> attachments from your computer system.
> #######################################################################################