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

-----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