Small quantum dots conjugated to nanobodies as immunofluorescence probes for nanometric microscopy

Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new ∼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed ∼2 nm precision.