Rather big gold particles (about 50 nm in diameter) were considered to be of optimal size for immunogold SEM, especially for overview examinations at lower magnifications. Smaller gold particles were not clearly visible in conventional microscopes. However, Walther et al.50 and Walther and Müller51 used an optimized BEI detector and showed that 5 to 15 nm gold particles can be unambiguously detected by SEM. The invention of silver enhancement (autometallography; AMG) helped to further improve the immunogold-SEM method. AMG can enhance the signal by precisely increasing the size of very small gold particles (1-5 nm in diameter) to specific desirable sizes.4,10,45 Because of steric hindrance effects, the bigger colloidal gold particles have limited access to the target molecules and therefore can label fewer epitopes-antigenic sites than smaller gold particles.26,51 When the gold particle size is only 1 to 5 nm compared to larger ones, the labeling density is increased.20,21 Furthermore, the AMG-amplified gold particles, in comparison to their gold counterparts of similar size, can be better recognized and viewed with BEI.19,45

Recently, it was demonstrated that glycerol, which has a very low vapor pressure and sufficient electrical conductivity, can be used to substitute water in biological samples.13,37 The use of liquid substitution brings a further improvement for broad range of specimens. Such samples infiltrated

with glycerol can be observed directly in the SEM without any drying. Due to the inherent electrical conductivity of glycerol, the samples can be examined without conductive coating, which improves the contrast of the marker. Furthermore, liquid substitution avoids the appearance of certain artificial structures, which occur during critical point drying or freeze drying typically, on specimens with a high water content, e.g., on mucilage layers. Glycerol infiltrated samples usually have smoother surfaces on which gold markers are clearly visible.