Improved detection of in situ hybridization by laser scanning confocal microscopy

Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques.