Chemical Drying: Biological Sample Preparation for Scanning Electron Microscopy (SEM)

This protocol is an excerpt from Koon et al., Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM), J. Vis. Exp. (2019).

1. Preparation and Fixation

1. Prepare Drosophila melanogaster (fruit fly).
   1. Anesthetize adult flies using 100% carbon dioxide. Place anesthetized adults (about 10 to 30 flies) in a small plastic screw cap vial or 1.5 mL centrifuge tube.
   2. Immerse anesthetized flies in 1 mL of fixative (1.25% glutaraldehyde, 0.1 M phosphate buffer pH 7.2) for 2 h (or overnight) at 4 °C. If the flies float to the surface of the fixative, add a few drops of 2.5% polyethylene glycol tert-octylphenyl ether to weaken the surface tension of the fixative allowing for total submersion of the tissue. Remove the fixative using a glass pipet.

2. Washing and Dehydration

1. Wash and dehydrate Drosophila melanogaster (fruit fly).
   1. Wash the fixed sample three times with 1 mL of 0.1 M phosphate buffer pH 7.2 at room temperature for 10 min in a 1.5 mL microcentrifuge tube. Remove each wash with a glass pipette, being careful not to remove the flies.
   2. Dehydrate the sample using a graded ethanol series, for 10 min in a volume of 1 mL in a microfuge tube. The ethanol concentrations are: 25%, 50%, 75%, 80%, 95%, 100%. Remove the ethanol with a glass pipette, being careful not to remove the flies.
   3. Retain the sample in the 1.5 mL microcentrifuge tube with just enough 100% ethanol to cover the sample before drying.

3. Drying

1. Perform chemical drying using hexamethyldisilazane (HMDS).
   1. Replace the 100% ethanol solution with a 1:2 solution of HMDS and 100% ethanol for 20 min. Replace the 1:2 solution with a 2:1 solution of HMDS and 100% ethanol for 20 min. Replace the 2:1 solution with 100% HMDS for 20 min. Repeat once.
      Note: HMDS is flammable and an acute toxin (dermal route). HMDS should be handled in a chemical fume hood using appropriate personal protective equipment including gloves, lab coat, and eye protection.
   2. Transfer the sample in HMDS, if in a 1.5 mL microcentrifuge tube, into a disposable aluminum weighing dish. Once in the aluminum weighing dish, replace the 100% HMDS with just enough fresh 100% HMDS to cover the sample.
   3. Transfer the sample to a plastic or glass non-vacuum desiccator with fresh desiccant (5-6 cm deep) and place into in a chemical fume hood. Alternatively, place the sample directly in a chemical fume hood to dry with a loose lid, such as a box, to prevent debris from falling on the sample. Allow the sample to dry for 12 to 24 h.

4. Mounting

1. Mount Drosophila melanogaster (fruit fly).
   1. Label the bottom of the aluminum mounting stub to indicate what is being placed on top.
      Place the dried flies in the desired position on adhesive or carbon adhesive tab secured to the top of a stub under a dissecting microscope with precision tweezers.
   2. Apply silver conductive adhesive, i.e., silver paint, around the outer edges of the stubs. Connect the silver paint to the flies using a toothpick to ensure conductivity. Do not allow the paint to touch the desired imaging area.
   3. Place the stubs in a stub holder box and place the open stub holder box in a desiccator. Allow the silver paint to dry at least 3 h or overnight for best results.