SEM of cells and tissues

Scanning Electron Microscopy (SEM) uses electron beams to gain information about the surface of cell or tissue specimens. Samples undergo chemical fixation to preserve native structure followed by a process of dehydration using critical point drying. Once dried samples are coated in a metal alloy and then imaged in the microscope. 

Critical Point Drying

Using the principles of supercritical drying, the dryer uses controlled conditions to replace all the solvent within the sample first with liquid carbon dioxide, then with gaseous carbon dioxide. The result is a sample that is completely dry while avoiding the damage caused by crossing the liquid/gas phase boundry. 

Example Method

The method outlined below was used for the preperation of Drosophila

 – Fix for 1-2hrs using a solution of 1.25% gultaraldehyde, 1% tannic acid in a phosphate          buffer. 

– Wash

– Postfix using 1% osmium tetroxide in cacodylate buffer. 

– Wash

– Dehydrate, using graded alcohol steps until 100% is reached. 

– Critical point dry

– Mount samples, using carbon glue and carbon pads to reduce charge on the sample. 

– Metal coat, using gold-palladium. 

 

 

Nature Neuroscience cover

Drosophila eye, prepared using SEM preperation techniques.