The phase shift given by 500nm of ice would be ~1/3 lambda, which is
easily measurable.  The tricky bit is keeping it frozen yet stopping it
getting thicker, and I really don't know how you'll do that - there's
obviously going to be a bit of engineering involved.  

What you really need is a quantitative interference microscope like the
old Zeiss Jena Interphako, which is designed for just such measurements.
There must be some modern equivalent - or you could probably make us an
offer for ours.  Nobody has touched it since I retired.

                                     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 Carol Heckman
Sent: Tuesday, 27 October 2009 3:53 AM
To: [log in to unmask]
Subject: Re: Quantification of phase contrast images

Ralf-
There is a reference to enhanced phase contrast in a recent paper.  This
might help, as the phase retardation introduced by water may be quite
small.  You can find it (open source) by searching Vasiliev and
"enhanced phase contrast".
Carol
Center for Microscopy & Microanalysis
Bowling Green State University
________________________________________
From: Confocal Microscopy List [[log in to unmask]] On
Behalf Of Ralf Zenke [[log in to unmask]]
Sent: Monday, October 26, 2009 6:12 AM
To: [log in to unmask]
Subject: Quantification of phase contrast images

Dear list,

For pre-observation of samples for electron microscopy we need to
measure the thickness of amorphous ice (~500nm) on thin (~10nm) carbon
meshes. We would like to use phase contrast (I think DIC could not be
quantified in that experiment because of the homogeneous thickness of
the ice layer).
Is it possible to get reliable thickness values (accuracy of ca. 50nm)
out of the gray values of the camera? Reference images without ice
layers can easily be aquired. Are there any effects that might corrupt
the measurements? I think it's at least neccessary to have a narrowband
light source.

Thanks to anyone who can give input on this!

Nice regards,

Ralf


Ralf Zenke

Max Planck Institute of Biochemistry
Core Facility
Am Klopferspitz 18
DE-82152 Martinsried near Munich
GERMANY
Phone: (+49) (89) 8578 3798
Fax: (+49) (89) 8578 2847
www.biochem.mpg.de<http://www.biochem.mpg.de>

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