Table 1.

Common technical problems with TEM and strategies for improvement.

Technical problem	Impact on image quality or analysis	Strategies for improvement
Under fixation of tissue	Tissue distortion and poor-quality staining	Make sure tissue is correct dimensions to allow for appropriate infiltration of fixative. Ensure appropriate volume to sample ratio. Fix tissues rapidly.
Over fixation of tissue	Tissue distortion and poor-quality staining Tissue will become brittle and hard to process	Reduce time that tissue is in fixative. Be sure that tissue is stored at appropriate temperature
Chemical fixation-induced sample contamination	Poor-quality Images	Use freshly made fixative solutions. Store fixatives correctly. Use caution and good laboratory techniques when using to avoid introduction of contaminants.
Inadequate washing	Speckled areas on tissue	Clean bottles used to store Osmium tetroxide with H2O2 and add some H2O2 to help prevent reduction of osmium tetroxide by glutaraldehyde.
Formation of precipitates (due to uranyl acetate and lead citrate)	Small black dots and speckled areas on tissue	Check pH of solutions. The pH of the uranyl acetate containing solution may need to be adjusted. Do not breathe on grids. Dry grids thoroughly. Limit light exposure for Uranyl acetate.
Inadequate resin filtration	Tears and holes in tissue	Make sure to perform solvent substitution. Resin needs to be mixed well with no bubbles. Allow appropriate time for epoxy resin infiltration and polymerization.
Toluidine blue staining	Too dark or too light	Too dark—rinse with water Too light—stain with toluidine blue longer
Imaging sections with high beam intensity	Tissue will break and curl up leaving the grid unusable	Set beam at lower intensities and slowly increase when necessary.
Image artifacts mistaken for real changes	Will lead to generation of inaccurate analyses and findings.	Learn and know how to identify common TEM artifacts. Work alongside a trained pathologist.
Analyses performed on a small number of images	May not reflect an accurate representation of what is occurring in specific tissue sample.	Take a minimum of 10–20 images per magnification of interest for analysis
Using both longitudinal and cross-sectional images for analyses	May not reflect an accurate representation of what is occurring in specific tissue sample.	Be sure to take images in longitudinal plane (unless looking at fiber changes) for analyses. Tissue pieces subjected to TEM must be representative of the sample as a whole.