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.
Department of Physics, ‡Center for the Physics of Living Cells, and §Center for Biophysics and Computational Biology, ∥Department of Bioengineering, and ⊥Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Yong
Y
Wang
En
E
Cai
Tobias
T
Rosenkranz
Pinghua
P
Ge
Kai Wen
KW
Teng
Sung Jun
SJ
Lim
Andrew M
AM
Smith
Hee Jung
HJ
Chung
Frederick
F
Sachs
William N
WN
Green
Philip
P
Gottlieb
Paul R
PR
Selvin
D000465:Algorithms; D002462:Cell Membrane / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D007473:Ion Channels / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016547:Kinesins / Q000032:analysis / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry; D009474:Neurons / Q000378:metabolism; D045663:Quantum Dots / Q000737:chemistry*; D018091:Receptors, AMPA / Q000032:analysis / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry; D061905:Single-Domain Antibodies / Q000737:chemistry*
D007473:Ion Channels; D015335:Molecular Probes; C515204:PIEZO1 protein, human; D018091:Receptors, AMPA; D057127:Single-Chain Antibodies; D061905:Single-Domain Antibodies; D049452:Green Fluorescent Proteins; D016547:Kinesins