A technical note on quantum dots for multi-color fluorescence in situ hybridization.

Quantum dots (Qdots) are semiconductor nanocrystals, which are photo-stable, show bright fluorescence with narrow, symmetric emission spectra and are available in multiple resolvable colors. We established a FISH protocol for the simultaneous visualization of up to 6 different DNA probes differentially labeled with Qdots and with conventional organic fluorochromes. Using a Leica SP5 laser scanning confocal microscope for image capture, we tested various combinations of hapten-labeled probes detected with streptavidin-Qdot525, sheep anti-digoxigenin-Qdot605, rat anti-dinitrophenyl-Qdot655 and goat anti-mouse-Qdot655, respectively, together with FITC-dUTP-, Cy3-dUTP- and Texas Red-dUTP-labeled probes. We further demonstrate that Qdots are suitable for imaging of FISH probes using 4Pi microscopy, which promises to push the resolution limits of light microscopy to 100 nanometers or less when applying a deconvolution algorithm, but requires the use of highly photo-stable fluors.