Table 1.

Troubleshooting guide for specimen preparation

Problem	Possible Cause	Possible Remedy
Structures appear shrunken	Improper fixation	Alter strength or type of fixative
	Hypertonic buffer	Adjust salt concentration
Excessive holes in membranes	Inadequate dehydration	Start dehydration series with lower concentration of ethanol or acetone
	Specimen exposed to air during fluid exchanges	Avoid unnecessary exposure to air.
		Avoid complete removal of solutions during exchange, do more exchanges
	Inadequate exchange in critical point dryer	Do more and extended exchanges of fluids in the CPD
	Put under vacuum before completely dried	Allow specimen to dry overnight before placing under vacuum
Debris on specimen	Incomplete washing with buffer	Additional wash steps prior to first fixations may be necessary
		Use of mild detergents or other reagents may be required
Capsular material appears sloughed off in background	Inadequate fixation	Additives may be required for stabilization, i.e. alcian blue or ruthenium red.
		Adjust pH or buffer appropriately
Specimen moves while imaging	Poor contact with mounting substrate	Add conductive paint around specimen to “seal the cracks”
Not enough material adhering to substrate	Concentration of specimen too low	Allow longer settling time before fixation
		Increase initial concentration
	Poor adherence properties	Pre-treat mounting substrate with adhesive
Excessive charging of sample (SeeTable 4)	Poor contact of specimen to mount	Improve contact by more firm placement on conductive adhesive, and coat with conductive paint if appropriate
	Inadequate metal coating	Increase thickness of metal coating
	Inadequate bulk conductivity	Increase bulk conductivity through OTOTO
Fine detail seems obscured And/or surface appears “chunky”	Excess metal applied during sputter coating	Apply thinner layer of metal
		Use finer grain or lower atomic number element