SPI-Chem™ Hexamethyldisilazane (HMDS)
More information about its use
This article was originally published in the May 1997 issue of
MICROSCOPY TODAY,
97-4, p16 (1997) and is presented here with permission.
Mr. Philip Oshel
Technical Editor
MICROSCOPY TODAY
PO Box 620122
Middleton, WI 53562
Ph: 1-(608) 836-1970
FAX:1-(608) 836-1969
HMDS and Specimen Drying for SEM
Hexamethyldisilazane (HMDS) is an excellent method of chemical drying of
hydrated specimens. There are several variables involved in its use, the most
easily controlled being the number of transitional steps from 100% ethanol
(EtOH) to 100% HMDS and the drying temperature.
Fixation and dehydration are the same for both HMDS and CPD. Once the
specimen is in the final 100% ethanol, it must then be transferred to 100%
HMDS through a graded series of ethanol-HMDS mixtures. This can follow one
of four basic paths:
Ratio absEtOH: HMDS starting from 100% EtOH going to 100% HMDS
1)100% E => 1:1=> 100% H
2)100% E => 2:1=> 1:2=> 100% H
3)100% E => 2:1=> 1:1=> 1:2=> 100% H
4)100% E => 3:1=> 1:1=> 1:3=> 100% H
(Extra gradations may be added as needed, for instance between the 3:1 - 1:1
and 1:1 - 1:3 steps)
After the final transitional step, make 3 changes in HMDS (the last two
steps can sometimes be skipped). Dry from the last 100% HMDS step, or
exchange with new 100% HMDS one final time then dry. There should be just
enough HMDS in the container to cover the specimen, any more just increases
the drying time.
Time in these steps will usually be the same as that used in the final 100%
EtOH steps. However, the time can apparently be extended will little ill
effect on the sample. Incomplete transition from EtOH to HMDS is a worse
source of problems than extended time in HMDS.
Choice of steps is basically determined by sample density, and the
permeability (to HMDS and ethanol) of the least permeable structures in the
sample. Microorganisms can usually be done with the first series, animal
tissues need the second or fourth series, and plants require the fourth
series, or even more gradations because of their cell walls.
Drying is done at either:
25º C = room temperature 8 hr => overnight
37º C }
45º C }1=>4hr
60º C }
(Drying time is determined by both temperature and volume of fluid.)
The higher the temperature, the shorter the drying time, but the quality of
results may also vary. Microscopic unicellular algae did best at 60º C, fish
skin at room temperature, bacteria equally well at room temperature and 60º C.
HMDS may have a significant time advantage over CPD. If more specimens are
being processed that can fit in the CPD chamber, then the times in the
transitional fluids and for drying will be much less than the time necessary
for CPD. The greater the number of samples that can be batch processed, the
greater the time advantage for HMDS.
HMDS has another advantage: if you can find containers that seal tightly
enough (HMDS likes to evaporate given any chance at all), samples can be
collected in the field, processed to 100% HMDS, then stored in vials filled
with HMDS and transported long distances from remote sites - like from
Antarctica to Chicago, Illinois. The samples are protected by the fluid,
and at least for some specimens, the extended time in HMDS produces fewer
artifacts than occur in specimens stored in fixatives or alcohol. This
procedure was used with fish lateral lines and their neuromast end organs,
on pieces of fish skin. Dried specimens are of course fragile.
HMDS is not the cure-all for specimen drying. It can introduce it's own
distorting artifacts and some samples, biological ones in particular,
still shrink after drying as they do with CPD or freeze-drying. Some
specimens do poorly when dried from HMDS. Plant tissues in particular
may not do well, and may be better off dried by CPD. Also, if the specimen
is going to be studied for elements that are labile, or in solution,
standard fixation and dehydration methods won't work. Cryo techniques must
be used for these specimens, and if the specimen is to be examined in an
unfrozen state, for example to look at structures and elements within
cavities that would be obscured by ice if left hydrated, then the specimen
must be dried by freeze-drying methods.
All of this information is empirical. Theoretical explanations and any
modifications for particular samples are welcome!
A final note: HMDS must be used in a flume hood! A sniff of it will clear
the sinuses back to the foramen magnum.
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