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