I think you are probably right but .... why would you do this rather
than just pull the slider out?
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Rietdorf, Jens
Sent: Sunday, 11 October 2009 6:00 AM
To: [log in to unmask]
Subject: Re: PSF with DIC
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
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