Cutting crossover

A mesenchymal cell labeled with three dye-conjugated antibodies and a nuclear stain. The top left image is a composite of the four markers.

Courtesy of Matthias Schieker, University of Münich

User: Matthias Schieker, University of Munich, Germany

The project: Identifying mesenchymal stem cells by labeling as many as seven characteristic proteins coexpressed on single cells.

The problem: Organic dye emission spectra overlap so that colors become mixed together.

The solution: Schieker began the project before quantum dots were available, and with the group's protocols working well, continues to use dyes (Dianova, Invitrogen). Appropriate filters for each dye are essential for separating the light to be detected from the excess light that overlaps with other dyes, Schieker says. To resolve small differences (10 nm) in wavelength, he uses spectral deconvolution, a software tool that extracts distinct signals from a composite image (see "Rendering images in 3-D," The Scientist, 18(8):40, 2004). According to Stuart Sealfon of Mount Sinai School of Medicine in New York, spectral deconvolution is necessary for resolving four or more dyes. The latest generation of confocal microscopes has built-in deconvolution software.