Posted by
Geoffrey Guttmann on
URL: http://confocal-microscopy-list.275.s1.nabble.com/water-window-x-ray-microscopy-tp5675290p5681492.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Jim and Otman and others with an interest in SXM
As one who did their dissertation on soft x-ray microscopy (SXM) many years
ago, I realized that confocal microscopy was much more effective at
recording changes in living biological samples and seeing what may occur
when prodded by some external stimulus or environment. Yes, EM and SXM are
very limited in that they can only really look at dead tissues or cells.
I did do some experiments to image living biological cells using a technique
called soft x-ray contact microscopy (SXCM), which is akin to the
semiconductor techniques used to make the chips used in computers. I used
both a x-ray C target tube source using supposedly only C x-ray lines and a
laser plasma x-ray source. The static system such as the C target x-ray tube
source killed the biological sample after a short period of time and didn't
make much of a useful image on the resist. I did get some images but they
were rather questionable. To solve this problem, I used some rather hearty
cell samples. The cells ended drying up and what was left was a fine
cytoskeletal framework with maybe a remnant of the cell membrane. The hazard
with doing SXCM is the resist must be read by another microscope, in this
case the SEM. When I realized I couldn't really image a living biological
cell with a x-ray C target tube source, I moved to using a laser plasma
source. Problem, at the time I did this work there were not many laser
plasma sources available. I did find a friend and colleague to help me out
and I managed to image a number of living cell samples using the laser
plasma source. There were a number of trials and tribulations until I got a
protocol that worked. Even then the living cell was not alive once the laser
plasma source had been fired. Thus one could not go back and look at the
cell again. I was able to capture the cells in movement and also during
mitosis. To verify mitosis however would have required that I kill the cell
and inject an antibody that could carry gold to the microtubules found in
the spindle fibers. Gold would be necessary to create contrast within the
cell. Essentially one creates a circular argument to justify doing these
experiments, when other light microscopy techniques may work better.
In short, soft x-ray microscopy is a technique in search of a problem to
solve. Biological tissues and cells are not that type of problem that can be
resolved by SXM very easily. Confocal microscopy can do a much better job in
this case. I have seen a number of physics tools being developed for SXM but
none have made an impression on me in the last twenty years. There are a
number of issues that need to be solved. One of these is radiation dose,
which is extremely high in SXM and SXCM, and will have a major effect on the
tissues or cells. Radiation dose is also a concern in doing transmission
x-ray microscopy because the question is, "Is the cell responding to the
radiation or to the environment acting as a stimulus?" Another issue is the
necessity to use an EM to view the resist in SXCM.
So, what biological issue can be resolved with SXM or SXCM? Well, one can
look at the botanicals or plant cells. Plant cells can handle high radiation
doses to a point before they die and they may not have much movement or be
static.
Cheers from someone who was converted from soft x-ray to confocal microscopy.
Geoff Guttmann, Ph. D. (Univ. of California, Berkeley 1989)
Professor of Anatomy
The Commonwealth Medical College
Scranton, PA 18509