Image courtesy of J. W. Lichtman, Washington University School of Medicine, St Louis, Missouri, USA.

The ability to selectively label tissue has been a great asset for the analysis of the architecture and connectivity of the brain. Although there has been rapid progress in recent years, particularly with the advent of green fluorescent protein (GFP) and similar compounds that can be used to label cells in transgenic animals, these approaches have some limitations. For example, although one can use plasmids encoding the GFP gene to label individual cells in slices or cultures, the expression of the gene (and therefore the labelling of the cell) may take up to six hours after transfection. This delay between transfection and labelling can limit the utility of this approach in some studies. Moreover, the specificity of the gene-gun approach that is used to fire plasmid-coated beads into the tissue can mean that high density labelling is achieved at the expense of tissue damage. One alternative is to use lipophilic, carbocyanine dyes, although it is technically difficult to label individual cells with this technique.

A paper published in the August issue of Neuron by Gan and colleagues describes an ingenious, hybrid approach. Their technique (DiOlistic labelling) also uses particle-mediated ballistic delivery by a gene gun but this time employs lipophilic dyes such as DiO, DiI and DiD to provide the label. By coating beads with different dye combinations, Gan and colleagues were able to show that living or fixed tissue could be rapidly labelled at high densities. Membrane filters placed between the gene gun and the tissue limited particle clustering and tissue damage. In live tissue, dendritic trees and glial cells appeared to be fully labelled within five minutes of particle contact. One of the exciting aspects of this study was that different neurons could be labelled with different colours either by using individual lipophilic dyes or combinations thereof. So by altering the relative quantities of different dyes up to seven different easily distinguishable colours were visualized, with the possibility of many more. Although this approach does have some limitations in certain situations, the description of the multicolour labelling DiOlistic technique may be a valuable addition to the armoury of the neuroanatomist.