Micromapping of the human brain: Three‐dimensional imaging of immunofluorescence and dendritic morphology using dual‐channel confocal laser scanning microscopy

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Abstract
A strategy for investigating neuronal networks at the microscopic level is described. The Lucifer Yellow microinjection technique was combined with immunofluorescence on human brain material, which was then studied in a confocal laser scanning microscope with dual‐channel scanning and equipped with an argon/krypton laser. The three‐dimensional architecture of the Lucifer Yellow‐injected neurons was investigated after transfer of the scanned frames in file format to a Silicon Graphics IRIS computer, using VoxelView software from Vital Images Inc. Microinjection of Lucifer Yellow revealed the dendritic morphology of various types of cells in different brain areas. Indirect immunofluorescence, with Texas Red as the secondary label, was used to determine the distribution of various categories of macromolecules (enzymes, receptor protein, and synaptic vesicle proteins) in the brain slices. We used single‐ as well as dual‐channel confocal laser scanning microscopy for imaging these double‐stained fluorescent specimens. Using these techniques in combination, we have created and saved three‐dimensional confocal images of detailed morphology (axons and dendrites with spines and varicosities) of individual cells, together with the localization of immunofluorescence. These three‐dimensional confocal images will be collected in a database for probable future use in human brain mapping.