Overview of small monovalent QDs, and accessibility of sQDs to synapses. (a) QDs are typically targeted to cell-surface proteins with antibodies (left), but streptavidin targeting reduces linkage size and increases stability. We decreased QD size by minimizing the passivating layer and reduced cross-linking by conjugating mSA. Finally we purified compact QDs (sQDs) bound to a single copy of mSA. (b) Fluorescence images of hippocampal neurons transfected with plasmids encoding AP-GluR2, BirA-ER and the post-synaptic marker Homer-GFP. Biotinylated APGluR2 was labeled at the cell surface with sQD-mSA₁, commercial QD605-streptavidin, or mSA- Alexa Fluor 568 and imaged live. Yellow shows the overlap between red QD- or Alexa-labeled GluR2, and green Homer puncta. Scale bar, 1 μm. Error, ± 1 s.d. (n = 6).

Monovalent sQDs reduce EphA3 clustering and internalization. (a) CHO cells expressing AP-EphA3 were biotinylated and labeled with sQD-mSA₁ or multivalent sQDs for 14 min at 37 °C and were imaged live (left). YFP was used as a transfection marker and illustrates that sQD labeling is specific for transfected cells (middle). Differential interference contrast (DIC) images are shown on the right. (b) CHO cells expressing AP-EphA3 were biotinylated, incubated with sQD-mSA₁ or multivalent sQDs for 14 min at 37 °C, and imaged before and after quenching of surface sQDs with trypan blue. Blue fluorescent protein (BFP) was used as a transfection marker. The percentage internalized sQDs is given as the mean ± 1 s.d. (n ≥ 4). Scale bars, 10 μm.

Generation and characterization of monovalent sQDs. (a) sQDs were incubated with mSA, separated on an agarose gel according to the number of mSA molecules attached, and visualized under UV light (left). Discrete bands corresponding to 0-3 copies of mSA per sQD are indicated. sQD-mSA₁ was purified from a bulk mixture of sQD-mSA conjugates (right). (b) To test valency by gel shift, sQD-mSA₁ was incubated with mono-biotinylated DNA and analyzed by agarose gel electrophoresis. In the reaction shown in the rightmost lane, sQD-mSA₁ was pre-blocked with free biotin, to test the specificity of QD-DNA binding. QD emission is colored green, DNA is colored red, and yellow shows the overlap. (c) To test valency by AFM, purified sQD-mSA₁ (left) or multivalent sQDs (right) were incubated with threefold excess mono-biotinylated DNA and imaged by AFM. The height profile along the black line is plotted in the right panel. Scale bar, 25 nm.