title: Single-molecule imaging of NGF axonal transport in microfluidic devices.
abstract: Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single-molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single-molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single-molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36-14 degrees C. A 10 degrees C decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single-molecule imaging of molecular processes in neurons.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Osakada
authors.forename: Marija
authors.initials: M
authors.lastname: Vrljic
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.forename: Harsha V
authors.initials: HV
authors.lastname: Mudrakola
authors.forename: Bianxiao
authors.initials: B
authors.lastname: Cui
mesh_terms: D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure*; D001693:Biological Transport, Active / Q000502:physiology; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D020932:Nerve Growth Factor / Q000378:metabolism*
chemical_list: D020932:Nerve Growth Factor