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Confocal microscopes have evolved over the past 25 years from the early stage scanning systems to a collection of sophisticated laser scanning systems designed for a range of biomedical applications. Major improvements to the photon efficiency of the instrumentation coupled with the development of novel fluorescent reporters have enabled multidimensional imaging of living cells and tissues.

Many technological advancements of the past decade have contributed to improvements in the photon efficiency of the confocal laser scanning microscope (CLSM). The resolution of images from the new generation of CLSMs is approaching that achieved by the microscope itself because of continued development in digital imaging methods, laser technology and the availability of brighter and more photostable fluorescent probes. Such advances have made possible novel experimental approaches for multiple label fluorescence, live cell imaging and multidimensional microscopy.

We present a simple means for triple-labeling biological specimens by immunofluorescence using a laser scanning confocal microscope for imaging with a krypton/argon laser as a light source. Three separate images of fluorescein-, lissamine rhodamine- and cyanine-5-labeled antibodies are collected and subsequently merged to form the triple-labeled image, which is displayed at full-image resolution (24 bit) on a second image processing system. The technique is illustrated using immunofluorescence localization of three segmentation proteins in Drosophila embryos.

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.