Heterogeneous Intracellular Trafficking Dynamics of Brain-Derived Neurotrophic Factor Complexes in the Neuronal Soma Revealed by Single Quantum Dot Tracking
(A) Sensitive detection of discrete QD-BDNF complexes in NG neurons (200 pM, left panel) compared to diffuse TrkB labeling with Ax488-BDNF (25 nM, right panel).Images are collapsed z-stacks (total cell height, 21–22 µm). QD-BDNFs are also detectable in single z-slice slices (yellow inset). (B) QD-BDNF complexes are detected inside single neuronal somata as single QDs. Representative fluorescent blinking profiles from two QD-BDNFs inside a neuron show square pulses of single ‘on-off’ QD blinking (red asterisks). x-axis = intensity, y-axis = time. (C) Highly-resolved spatial detection of QD-BDNFs accurately determines membrane vs. cytoplasmic location of QD-BDNF complexes in neurons. 3D models of location of single QD-BDNF complexes (green) with respect to Ax488-WGA-labelled membrane (magenta) are computationally processed from raw fluorescence data (inset, 2.7 µm thick z-stack projection taken at neuronal mid-section). (D) QD-BDNF complexes within neuronal somata can be tracked for extended time durations. Single QD-BDNF vs. diffuse Ax488-BDNF fuorescence inside a neuron over time (top). Average intensity as a function of excitation duration show quantitative comparison of extended fluorescence stability of QD625-BDNF vs. Ax488-BDNF (n = 10, middle). Under these extended recording sessions, corresponding DIC images of a representative neuron before and after 10 min of fluorescence excitation shows maintained morphological integrity (bottom). Scale bars: 20 µm (A), 10 µm (C, D).