Any adjustment of the analyzer will also shift the distortion in a  
manner depending on the implementation of the DIC in any particular  
microscope.  Each adjustment requiring then its own PSF.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
[log in to unmask]

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The box said "Requires WindowsXP or better", so I bought a Macintosh.
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On Oct 10, 2009, at 1:36 PM, James Pawley wrote:

>> Dear,
>>
>> though the prism does distort the image, I do not see how it would
>> reduce the NA, so basically the resolving power should be  
>> unaffected and
>> it would 'just' need a clever piece of de-distortion software to
>> restore. Has anyone of you tried to use a measured PSF to de-convolve
>> such a distorted image?
>>
>> Thanks, jens
>
>
> Certainly, you could do this (keeping in mind that the DIC  
> distortion of the PSF changes somewhat over the field of view). In  
> addition, you could not use the trick of rotational averaging that  
> allows one to reduce the statistical noise in the measured PSF. But  
> even a distorted PSF is still a PSF and can be used to deconvolve.
>
> But remember, a distorted PSF (which at the very least comes about  
> from having every point-object seem to be located in two partially- 
> overlapped positions) is bigger than an undistorted one. And losing  
> resolution in this way can never be good.
>
> Jim Pawley
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List  
>> [mailto:[log in to unmask]]
>> On Behalf Of Robert Carter
>> Sent: Saturday, October 10, 2009 12:05 PM
>> To: [log in to unmask]
>> Subject: Re: PSF with DIC
>>
>> I use our lab's Deltavision (IX-70 base) almost exclusively for the
>> live-cell
>> imaging.  I've already completed a good bit of hi-res imaging and  
>> fixed,
>> stained
>> cells before proceeding to live-cell, time-lapse experiments.
>>
>> The PSF is affected and resolution deteriorates when the DIC  
>> objective
>> prism
>> is left in the light path during epi-fluorescence imaging.  That  
>> said, I
>> leave the
>> objective prism removed for fixed-cell imaging because I only need  
>> the
>> epi-
>> fluorescence. It's in place for live-cell work though because DIC is
>> highly-
>> useful.
>>
>> For live-cell, compromises always have to be made to cut down on the
>> phototoxicity.  DIC is no exception.  I am imaging HPV plasmids bound
>> inducibly by a GFP fusion peptide.  Some rules I learned from the
>> instructors
>> and company reps at the 2002 Cold Spring Harbor course have served me
>> very
>> well and yielded great success.   1)Use pixel binning - live-cell  
>> is about observing the dynamics of the
>>
>>     biological process.  2x2 binning allows is more than sufficient  
>> to
>>     corroborate fixed cell images and allow for accurate reporting of
>> dynamic
>>     processes.
>>  2)During live-cell imaging, I collect the bright-field and  
>> fluorescent
>> image
>>    channels through the GFP emission filter.  The bright-field image
>> quality is
>>    just fine even though I'm not using the Analyser.  I might collect
>> eight or
>>    ten z-plane images per cell per time point.  I am eliminating
>> fifteen to
>>    nineteen emission filter changes in this instance.  This saves a  
>> lot
>> of time
>>    and keeps the cells happier for longer.
>>  3)Use higher-intensity illumination together with shorter exposure
>> times.  The
>>    imaging for each time point is completed sooner, cells have
>>    more "rest"/"recovery" time between time points, and they stay
>> healthy for
>>    much longer.  This is crucial, especially more mitotic cells.  I  
>> can
>> routinely
>>    image twelve to fifteen mitotic cells prior to the next time  
>> point's
>> imaging
>>    pass.
>>
>> I hate losing three-fourths of the signal so I take the objective  
>> prism
>> out and
>> safely stow it until I need DIC.  I had my bosses put a keycode  
>> access
>> lock on
>> our scope rooms.  Each user has his own code, and each time the user
>> enters
>> the room, a person-specific log entry is generated.  It's amazing how
>> much
>> less damage and destruction occurred once we implemented this policy.
>> Just
>> ensure that users are comfortable taking the multi-thousand dollar
>> objective
>> prism out when DIC isn't required.
>>
>> This is my first posting although I am a long-time subscriber to the
>> confocal
>> list.
>>
>> Hope this helps and isn't too redundant.
>>
>> Best luck,
>>
>> Rob Carter
>> Lab of Tom Broker and Louise Chow at UAB
>> 505 MCLM Bldg
>> Birmingham, AL 35294
>> 205-975-8304
>
>
> -- 
> James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726  
> Phone: 608-238-3953