Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

This protocol focuses on two chemical drying methods, Hexamethyldisilazane and T-butyl alcohol and their application imaging both prokaryotic and eukaryotic organisms using SEM. The main advantage of this technique is that it does not require the use of specialized drying equipment such as a critical point dryer to prepare samples for analysis. This protocol describes the methods for using chemical dehydration and SEM analysis of three types of organisms but would be broadly applicable to examining many organismal and tissue types.

Demonstrating the procedure will be Madison Koon, an undergraduate student from my laboratory. To start, prepare cyanobacteria by overnight fixation. Then, transfer them into a 10 mL filtration rig containing a polycarbonate 25 mL filter.

Seal the sidearm and funnel of the filtration rig with rubber stoppers to contain the culture in the funnel. After removing both stoppers, remove the fixative by gentle vacuuming on the filtration flask. To prepare single-cell algae euglenoids, fix them by adding 3 drops of 4%osmium tetroxide directly to the culture that has been previously transferred into a 10 mL filtration rig.

After a 30 minute incubation, remove both stoppers and remove the fixative by gentle vacuuming on the filtration flask. Wash the fixed euglenoids three times with 5 mL of 0.1 M phosphate buffer pH 7.0 at room temperature for ten minutes each while keeping the flask and funnel closed with the stoppers to hold the wash. After removing both stoppers, remove each wash by gentle vacuum on the filtration flask.

To dehydrate the sample while maintaining continuous immersion, use a graded ethanol series for ten minutes in a volume of 5 mL in the funnel while keeping the flask and funnel closed with the stoppers to contain the ethanol in the funnel. After removing both stoppers gently vacuum on the filtration flask to remove ethanol. Transfer the sample to a disposable aluminum weighing dish containing just enough 100%ethanol to cover the sample.

To perform chemical drying of the euglenoids using TBA, first replace the 100%ethanol solution in the aluminum dish with a 1 to 1 solution of TBA and 100%ethanol for twenty minutes. Then, replace the 1 to 1 solution with 100%TBA for twenty minutes and repeat the process twice. Remove TBA and replace with just enough fresh 100%TBA to cover the sample.

Place the dish at four degrees celsius for ten minutes to freeze the TBA. Transfer the sample to a vacuum desiccator with frozen gel packs to keep TBA frozen. Use a rotary pump to evacuate and maintain vacuum to allow the sample to dry by vacuum sublimation of the frozen TBA for at least three hours.

To mount cyanobacteria in euglenoids, label the bottom of a 25 mm aluminum mounting stub to indicate what is being placed on top. Then place each filter on a carbon adhesive tab secured to the top of the stub. To prepare Drosophila melanogaster, immerse anesthetized flies in 1 mL of fixative for two hours at four degrees celsius, making sure they are completely submerged.

Use a glass pipette to remove the fixative. Wash the fixed Drosophila sample in a 1.5 mL microcentrifuge tube three times with 1 mL of 0.1 M phosphate buffer, pH 7.2 at room temperature for ten 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 mL using a graded ethanol series for ten minutes each. Use a glass pipette to remove each wash, being careful not to remove the sample. Keep the sample in the 1.5 mL 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 twenty minutes. Then replace the solution with a 2 to 1 solution of HMDS and 100%ethanol for twenty minutes. Finally, replace this solution with 100%HMDS for twenty minutes and then repeat the process once.

Transfer the sample which is in a 1.5 mL micro centrifuge 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 a 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 stubs.

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. To prepare the sputter coating apparatus, perform 4 to 5 flushes of argon gas.

Depending on the samples to be coated, set the timer to different settings as described in the manuscript. Sputter coat the stubs following the manufacturers instructions. Finally, image samples using a scanning electron microscope that includes a secondary electron detector with settings that depend on the sample used.

Successful preparation of cyanobacteria for SEM using this protocol yielded images where details of the cell, including shape and texture of the surface are visible as well as a sheath of mucilage and projections from the cells. SEM image of a filamentous freshwater species from Colorado shows a visible sheath of mucilage. SEM image of a freshwater bacteria from Ohio reveals individual cells which sometimes form colonies held together by a thin layer of mucilage.

Filament-like protrusions extend from the individual cells. Images of unknown filamentous species from a high salinity estuary in Texas coastal waters show individual and dividing cells. SEM was used to visualize the pellicle strips of two different Euglenoid species.

When comparing Monomorphina and Aenigmatica with Monomorphina pseudo pyrum, the visible helical arrangement of the pellicle strips that emerge at the posterior end to form a tail, aid in phylogeny to termination. SEM images of normal drosophila eyes show an ordered array of bristles and lenses but expression of proteins associated with Alzheimer's disease create the rough eye phenotype. Genes that suppress or enhance the rough eye phenotype may play a physiological role in disease progression.

Here are examples of potential pit falls to avoid, including an example of the collapsed structure of the eye from improper drying technique and electron charging from insufficient sputter coding that appears during image acquisition. The protocols described here utilized organisms that very greatly with respected complicity, yet all are amenable to SEM analysis to gather useful, morphological information that is critical for scientific discoveries spanning many sub disciplines of biology. Don't forget that working with T-Butyl alcohol, Hexamethyldisilazane, and osmium tetroxide can be hazardous and the appropriate safety measures should always be observed to protect users, particularly students who will be handling these chemicals.