Chemical Drying: Biological Sample Preparation for Scanning Electron Microscopy (SEM)
Transcript
– To begin, start by dehydrating and drying a fixed sample to reduce sample distortion caused by the removal of gases and liquid water at the reduced operating pressure of conventional scanning electron microscopes, SEMs.
To dehydrate the sample, wash it with increasing concentrations of ethanol in water until 100% ethanol is reached. Then repeat this process with a chemical drying agent in ethanol until the sample is washed with pure drying agent. Transfer the sample and drying agent to an evaporating dish. Cover it with minimal additional drying agent and let it dry in a fume hood overnight.
Next, mount the prepared sample on the top of a microscope stub using carbon adhesive tape. Finally, apply silver paint to the edges of the stub, then from the edge of the stub to each of the samples, to reduce the buildup of excess electrons, which could result in undesirable image artifacts.
In this experiment, fruit flies, Drosophila melanogaster, are prepared for SEM analysis using the drying agent HMDS.
– To prepare Drosophila melanogaster, immerse anesthetized flies in 1 milliliter of fixitive for 2 hours at 4 degrees Celsius, making sure they are completely submerged.
Use a glass pipette to remove the fixitive. Wash the fixed Drosophila sample in a 1.5 milliliter microcentrifuge tube three times with 1 milliliter of 0.1 molar phosphate buffer pH 7.2 at room temperature for 10 minutes. Use a glass pipette to remove each wash, being careful not to remove the sample.
Then dehydrate the sample in a microfuge tube, in a volume of 1 milliliter, using a graded ethanol series for 10 minutes each. Use a glass pipette to remove each wash, being careful not to remove the sample. Keep the sample in the 1.5 milliliter microcentrifuge tube with just enough 100% ethanol to cover it.
To perform chemical drying of the flies using HMDS, first, replace the 100% ethanol solution with a 1 to 2 solution of HMDS and 100% ethanol for 20 minutes. Then replace the solution with a 2 to 1 solution of HMDS and 100% ethanol for 20 minutes. Finally, replace this solution with 100% HMDS for 20 minutes, and then repeat the process once.
Transfer the sample, which is in a 1.5 milliliter microcentrifuge tube and HMDS, into a disposable aluminum weighing dish. Replace the 100% HMDS with just enough fresh 100% HMDS to cover the sample. Place the sample within the aluminum dish directly in a chemical fume hood to dry with a loose lid, such as a box, to prevent debris from falling on the sample, and allow it to dry for 12 to 24 hours.
To mount Drosophila label the bottom of an aluminum mounting stub. Use precision tweezers to place the dried flies in the desired position on a carbon adhesive tab secured to the top of a stub under a dissecting microscope. Apply silver conductive adhesive around the outer edges of the stubbs.
Use a toothpick to connect the silver paint to the flies, ensuring conductivity, making sure that the paint doesn’t touch the desired imaging area. Place the stubs in a stub holder box. And then place the open stub holder box in a desiccator and allow the silver paint to dry for at least three hours.
