Our problem is that we do marine biology and most specimens come with a layer of salt water or, if dissected, then a film of body fluid. What happens to the surface layer - I know from cryo that after sublimation we are left with a driedsalt layer which can be unhelpful! Sometimes I have been known to rinse specimens in fresh water - that helps!

Any comments would be welcomed

Keith Ryan
e-mail: k.ryan@pml.ac.uk

Dear Keith,
I have not had experience with ESEM's, but I have used an environ- mental chamber on the high-voltage TEM. I don't know what is commercially available, but I can tell you a few relevant details about our system.

Ours is a differentially-pumped chamber, which has a reservoir of water (or, potentially, other liquids) and four apertures. The inner two separate the inner chamber from the differential pumping region, and the outer two separate that region from the high vacuum of the column. Water vapor flows through the inner pair of apertures and is pumped away, and the beam passes through all four apertures. The aperture size was selected to allow steady-state operation--water from the reservoir evaporates at the same rate as vapor flows out--and the pressure in the chamber is the vapor pressure of water at the operating temperature.

The chamber stays at ~100% humidity; we tested this by finding dif- fraction patterns from catalase, which is irreversably disordered at < 95% humidity. We also cultured some PTK1 cells on gold grids, removed them from the dish, rinsed them with fresh water, blotted the excess until only a thin film of water remained (~10 A) and put them into the chamber.

Some were left in the chamber for 1/2 hour and then removed and replaced into the culture dish without being exposed to the beam, and others were similarly left in the chamber then subjected to the beam and photographed. Only one edge of these grids was exposed to the beam, but there was x-ray exposure to the entire grid. Photos were taken with Lo Dose film, so there was less exposure than for the usual imaging conditions. Subsequently, these grids were also replaced into a culture dish. At least some of the cells on each of the grids were still alive after these treat- ments--of course, none of the irradiated cells on the exposed edges of the grids survived.

The answer to where the water on your specimens would go is that at steady state it should neither decrease from evaporation nor increase from condensation, if the vapor pressure at the reservoir is the same at that at the specimen. This may mean that you would have to add a solute to the reservoir to achieve the same vapor pressure as sea water. Your specimens may also be less tolerant of a fresh-water rinse. Good luck.

William Tivol
tivol@wadsworth.org

Dear Stephan,
Yes, there is probably a considerable local temperature increase, but this is minimized by the use of low-dose imaging. The local water vapor pressure probably doesn't change too much, since the water-vapor currents will keep the pressure uniform. One thing to remember is that there is no air--only water--in the set-up we use.

Under these conditions, there is neither net condensation nor evap- oration, so the thin aqueous film on wet specimens stays the same. The catalase ED shows that the order is preserved for at least 100 sec--the length of the exposure of the film--since the pattern is still visible after the picture is taken.

William Tivol
tivol@wadsworth.org