This protocol describes two different methods for dehydrating adult Drosophila in preparation for S.E.M.; For S.E.M. the water in a specimen must be removed without collapsing it by the surface tension forces which occur as the liquid evaporates. Air drying is unsatisfactory for adult Drosophila. In addition to providing better preservation, the two methods described below have the added advantage of rinsing the flies with an organic solvent and thus removing exterior dirt and grime. The first method requires the use of a critical point drying apparatus which most E.M. laboratories will have. The second protocol employs the use of a low surface tension solvent such as Freon 113.
1) Anesthetize the adult flies with CO2 or your favorite anesthetic unless of course you
are a cruel and sadistic person.
2) Place ten flies into 5 mls of 25% ethanol and incubate for 12-24 hrs at r.t.
3) Further dehydrate the flies through 50%, 75% and 2 x 100% ethanol as in step 2
above. Once in 100% ethanol the exterior morphology of the fly eye is stable for at
least one month.
FURTHER STEPS FOR CRICTICAL POINT DRYING
4) Crictical point dry ;the samples. (Critical point drying is a process of exchanging the
ethanol for liquid CO2 under high pressure. Once the ethanol has been replaced with
liquid CO2, the temperature of the critical point drier can be raised such that the CO2
becomes gaseous. The transition from liquid to gas at temperature X and pressure Y
is the critical point. The CO2 gas can then be vented to the atmosphere thus escaping
from the sample with very low surface tension and leaving the sample dry. Check
with your local E.M. person for details on how to safely use a critical point drier.
There is a good reason for calling these things bombs.)
FURTHER STEPS FOR LOW SURFACE TENSION SOLVENTS
4) Pass the samples through a graded series of ;Freon 113 in ethanol (25%, 50%, 75%, 2
x 100%) with 12-24 hr incubations in each step of the series at r.t. After the last
100% step, remove the Freon 113 and vacuum dry the samples. (Because of the
effects which fluorcarbons like Freon 113 have on the ozone layer you may want to
use an alternative low surface tension solvent. The solvent hexamethyldisilazane is
reported to be such an alternative solvent. The following reference claims that
dehydration through this solvent can give better results than critical point drying (J.
Microscopy 121: 185-189).
COMMON STEPS
5) The dried samples can be stored indefinitely under vacuum with dessicant.
6) The samples are then mounted onto S.E.M. stubs using T.V. tube coat (Ted Pella
Inc.). This adhesive is nice because it provides a good black background for
photography. Care should be taken to mount the samples with the appropriate
orientation so that the external structures of interest can be observed on the scope.
7) Sputter coat the samples with a 25-nm-thick coat of platinum;.
8) The samples are now ready for viewing and should be stored over dessicant.