Conductive staining in SEM with especial reference to tissue transparency

Abstract

Although the number of applications of conductive staining for SEM has recently increased investigations or discussions on the features of these methods are scanty, and so it could not be said that its features are sufficiently understood. In the present studies employing the tannin‐osmium method as the conductive stain, the authors compared it with an ion‐sputtering method that they use for metal coating. Besides the prevention of masking by the coating metal, there are other advantages to the conductive staining method. The appearance of the tissue “transparency”, which allows three‐dimensional observations of sub‐surface structures to be made. Any damage associated with metal coating can be avoided since the specimen itself is conductive. Hardening of specimens by the conductive stain enhances the resistance of structures to mechanical damage during preparation. Especially the tissue transparency produced by the conductive stain might suggest new possibilities for SEM.

Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

“…Concerning tannic acid, it reacts with osmium tetroxide and increases lipid retention, forming complexes that link to proteins and carbohydrates [ 90 ]. Consequently, their combined use enhanced extracellular matrix resistance to mechanical damage during sample preparation (specimen hardening, [ 91 , 92 , 93 , 94 ]). The sample acquires intrinsic conductive properties into the bulk, not only on the surface (as it happens with sputter coating), but enhanced image contrast without charging phenomena, and 3-D observation of sub-surface structures under higher voltages are allowed [ 95 ].…”

“…Tannic acid impregnation was used to harden Eps and to render the sample itself conductive without the appearance of charging phenomena [ 89 , 90 , 91 ]. Sputter coating allows only the surface to be conductive, so the combined use of OsO 4 -RR in the post-fixation step, followed by tannic acid impregnation, improved observation, allowing visualization of the sub-surface structures like bacterial cell embedded in the biofilm matrix [ 94 ]. Finally, to maintain the biofilm in a hydrated state, but to observe it under high vacuum and high voltage conditions, ionic liquid drop-casting was used.…”

Characterization of Scardovia wiggsiae Biofilm by Original Scanning Electron Microscopy Protocol

“…Consequently, they enhance extracellular matrix resistance to mechanical damage during preparation procedures, thanks to a sort of specimen hardening [41][42][43][44]. This method renders the sample itself conductive (not only its surface, as it happens with sputter coating), enhances the image contrast without charging phenomena and allows a three-dimensional observation of its sub-surface structures under higher voltages in comparison with VP-SEM [45].…”

“…TA increases lipid retention and binds to proteins and carbohydrates forming complexes; TA reacts with osmium tetroxide [40] and the consequences are enhanced contrast, specimens hardening and an enhancement of extracellular matrices' resistance to mechanical damage during preparation procedures [41][42][43][44]. This method renders the sample itself conductive without charging phenomena (not only the surface as it is with sputter coating), allowing the three-dimensional observations of its sub-surface structures combined with the use of higher voltages with respect to VP-SEM [45].…”