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