Scanning electron microscopy protocol
Rene Flores Clavo, Francisco Breno S. Teófilo
Abstract
This protocol briefly summarizes the basic steps of a scanning electron microscopy processing. The methods adopted for fixation, post-fixation, dehydration, drying in a critical point chamber, sputter coating, and the visualization and acquisition of images are described here.
Steps
MATERIAL SELECTION
1 mm2 samples were selected in the colony.
FIXATION
Samples were fixed in a solution of 2.5% volume 0.1Molarity (M) 7.3) , at Room temperature, for 4h 0m 0s.
WASHING AFTER FIXATION
Samples was then rinsed three times in 0.1Molarity (M) 7.3), at Room temperature , for 0h 20m 0s for each rinse.
POST-FIXATION
Samples was then post-fixed with 1% volume osmium tetroxide in 0.1Molarity (M)7.3, at Room temperature, for 1h 0m 0s, protected from ambient light.
WASHING AFTER POST-FIXATION
Samples was then rinsed three times in sodium 0.1Molarity (M) 7.3, at Room temperature, for 0h 20m 0s for each rinse.
DEHYDRATION
Samples were dehydrated with ascending acetone series, for 0h 20m 0s minutes each step - 30% volume; 50% volume, 70% volume, 90% volume, 100% volume (last one step, for three times).
CRITICAL POINT DRYING
Samples was then dried using the critical point method with CO2.
MOUTING ON THE STUBS
Samples was then placed on aluminum stubs.
SPUTTER-COATER
Samples was then coated with a layer of 30−40 nm gold using a Balzers SCD 050 sputter-coater.
OBSERVATIONS AND IMAGE ACQUISITION
Observations and photomicrograph aquisitions were obtained using a JEOL JSM 5800LV at 10 kV with SemAfore 5.21 software.
