Use of quantum dots for live cell imaging

Monitoring interactions within and among cells as they grow and differentiate is a key to understanding organismal development. Fluorescence microscopy is among the most widely used approaches for high-resolution, noninvasive imaging of live organisms(1,2), and organic fluorophores are the most commonly used tags for fluorescence-based imaging(3). Despite their considerable advantages in live cell imaging, organic fluorophores are subject to certain limitations. Fluorescent quantum dots (QDs) are inorganic fluorescent nanocrystals that overcome many of these limitations and provide a useful alternative for studies that require long-term and multicolor imaging of cellular and molecular interactions(4,5). For labeling specific cellular proteins, QDs must be conjugated to biomolecules that provide binding specificity. Bioconjugation approaches vary with the surface properties of the hydrophilic OD used. The mixed surface self-assembly approach is recommended for conjugating biomolecules to QDs capped with negatively charged dihydroxylipoic acid (DHLA) (Fig. 1). In this approach DHLA-capped Us are conjugated to proteins using positively charged adaptors(6-8), for example, a naturally charged protein (e.g., avidin(9)), a protein fused to positively charged leucine zipper peptide (zb)(8) or a protein fused to pentahistidine peptide (5 x His)(10). The use of avidin permits stable conjugation of the QDs to ligands, antibodies or other molecules that can be biotinylated, whereas the use of proteins fused to a positively charged peptide or oligohistidine peptide obviates the need for biotinylating the target molecule. This procedure describes the bioconjugation of QDs and specific labeling of both intracellular and cell-surface proteins(4,5). For generalized cellular labeling, QDs not conjugated to a specific biomolecule may be used; various strategies are presented in Box 1, Generalized Labeling of Live Cells.