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