Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/water-window-x-ray-microscopy-tp5675290p5678791.html
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>Hi ALL,
>
>Water window soft X-ray microscopy is used successfully at Synchrotron
>facilities to image bio samples. Water window is because water is
>transparent to soft X-rays and only carbon constituents of the cell are
>imaged. Could you please let me know how this techniques compares to other
>microscopy techniques for bio applications.
>
>Many thanks,
>Otman BENALI, PhD
>
>NANO-UV SAS, Paris/FRANCE
>
http://www.nanouv.com>
http://www.mcxi.euDear Otman,
After 50 years, x-ray microscopy remains a shimmering promise on the
fringes of biological microscopy.
I believe that it will always remain there.
Initially, the argument was the Abbe Limit of 200nm in light
microscopy that could only be overcome with shorter wavelengths.
Hence x-rays. The problem was that there were no sources bright
enough and no lenses. In the mean time, electron microscopy and later
cryo-electron microscopy filled the void, and filled it pretty well.
No matter, it always made it easier to get massive Congressional
appropriations for physics toys if one could hold out the promise of
improved biological imaging. So x-ray microscopy refused to die. Over
time, the sources got brighter and rudimentary lenses were developed.
Although these devices were very clever in themselves, their
performance parameters were vastly inferior to those of their
electron-optics counterparts (For instance, although a zone plate can
indeed focus about 10% of a parallel, monochromatic x-ray beam into a
small spot, the other 93% scatters all over the place.) There was
always progress and a new idea on the horizon, but if you will permit
a personal observation, these ideas were often pushed by people who
seemed to have very little understanding of what was already being
done in other fields of biological microscopy (I know, I went to some
of their meetings. In the early, they were still talking about EM at
the level it was practiced in the early 1960s).
As time went on it became obvious that both EM and x-ray microscopy
were not in the end limited by the lenses or the sources but by the
interaction of the beam with the specimen. In particular, both use
ionizing radiation that produced inelastic interactions that
deposited energy in the specimen and they did so at a high enough
energy that covalent bonds were broken. This became increasingly
important as the resolution improved (more damage to smaller area).
Looking at living specimens beyond normal LM resolution was a mirage.
(My definition is that you only look at living cells to see some
change. This means at least two images with some hope that the
damage created by making the first is so slight that the second image
has some interest. This excludes both EM and XM)
Once one understands this, the parameter of interest is how to get
the most information for the least damage and this leads us to the
details of the scattering and the contrast of the specimen. The full
description would require a short book but in short, EM has better
(cheaper, smaller more available) sources, better lenses (now they
have corrected the aberrations MUCH better) and the interactions have
higher intrinsic contrast (even in the fabled water window, where the
x-ray contrast is indeed better but the claimed resolution is
relatively worse) and far more of the interacting quanta can be
collected in the final data.
In short, be very careful not be be swept away by all the shiny
stainless steel and compare any results you see with other methods of
looking at dead tissue (like cryo-electron tomography or for living
cells: STED?).
And don't forget the time element. Most microscopy studies in biology
are limited first and foremost not by "resolution" by not looking at
enough specimens. There is a big difference between a scope down the
hall and one across the country.
Grumpy Jim Pawley
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James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726
Phone: 608-238-3953