Direct immunofluorescence of plant microtubules based on semiconductor nanocrystals

Fluorescence microscopy in combination with multiple, simultaneous labeling of biomolecules has been a key breakthrough in cell biology. However, the spatiotemporal resolution of this approach is limited by bleaching of the fluorescence label and illegitimate cross-reference of the label. CdSe-based semiconductor nanocrystals with their excellent bleaching stability would be an alternative to overcome this limitation. We therefore explored direct immunofluorescence based on nanocrystal-conjugated antibodies using plant microtubules as model. We compared two strategies of bioconjugation, covalent coupling of antitubulin antibodies to BSA-coated nanocrystals and covalent coupling to nanocrystals that were surrounded by functionalized silica shells. Both nanoparticle-antibody conjugates were used to follow the dynamic reorganization of microtubules through the cell cycle of a tobacco cell culture in double and triple staining with FITC as conventional fluorochrome and Hoechst 33258 as marker for mitotic duplication of DNA. BSA-coated nanocrystals visualized fluorescent dots that decorated the various arrays of microtubules. The specificity of the antibody was maintained after conjugation with the nanocrystals, and the antibodies correctly represented the dynamics of cell-cycle-dependent microtubular reorganization. However, this approach did not yield a contiguous signal. In contrast, silica-shelled nanocrystals visualized contiguous microtubules in the same pattern as found for the conventional fluorochrome FITC and thus can be used as labels for direct immunofluorescence in plant cells.