IMAGING OF RNA IN-SITU HYBRIDIZATION BY ATOMIC-FORCE MICROSCOPY

In this study we investigated the possibility of imaging internal cell ular molecules after cytochemical detection with atomic force microsco py (AFM). To this end, rat 9G and HeLa cells were hybridized with hapt enized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids w ere subsequently detected with a peroxidase-labelled antibody and visu alized with 3,3'-diaminobenzidine (DAB). The influence of various scan ning conditions on cell morphology and visibility of the signal was in vestigated, In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells we re kept in phosphate-buffered saline (PBS) and scanned under fluid aft er DAB development or dehydrated and subsequently scanned dry or subme rged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mo ck-hybridized cells was available. Results show that internal DAB prec ipitate can be detected by AFM, with the highest sensitivity in the ca se of dry cells, Although a relatively large amount of DAB had to be p recipitated inside the cell before it was visible by AFM, the resoluti on of AFM for imaging of RNA-in situ hybridization signals was slightl y better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects o n cellular structure. Therefore, scanning under fluid of previously de hydrated samples cannot be considered as a good representation of the situation before dehydration.