Second, the soft grease barrier is vulnerable to any mechanical disturbances when transporting the culture from the CO 2 incubator to the microscope stage. First, the vacuum grease barrier gives rise to huge fluorescence background, preventing any single molecule detection in its vicinity. While the Campenot chamber effectively separates distal axons from the cell body, it is technically challenging to combine with single molecule imaging due to a number of reasons. Our initial success of single-molecule observation of fluorescently labeled endosome relied on the use of compartmentalized Campenot chambers, that allow localized application of NGF to axonal termini 12, 13, to spatially filter out the strong background arising from the fluorescence molecules diffusing in solution or binding at the surface of axons. Large variations in transport patterns among NGF-containing endosomes call for quantitative characterization of individual behavior, obscured by ensemble averaging. Our recent report 11 of tracking NGF transport in axons revealed that the majority of endosomes contain single NGF dimer molecule and their movements are intrinsically heterogeneous. These endosomes carrying the NGF/TrkA complex together with an array of other signaling molecules are retrogradely transported to the cell body via a microtubule-based transport mechanism 9, 10. Following the binding of NGF to its TrkA receptor at the axon terminal, the ligand-receptor complex is endocytosed and trafficked to specialized endosomes 4, 8.

NGF is produced and released by target tissues to activate membrane receptors on the distal axons of innervating neurons. Therefore, it is of immense interest to understand axonal transport in great detail. Disruption of axonal transport often precedes the death of the cell body and is linked to many neurodegenerative diseases 7. NGF) derived from the target cells at the distal axon sites, which are essential for the survival and maintenance of neurons, are carried retrogradely by axonal transport to the cell body 4 – 6. As a result, neurons rely on active axonal transport for all the materials needed at synaptic terminals, including neurotransmitter receptors, synaptic proteins, ion channels, lipids, and mitochondria 3. Such long axons preclude effective diffusion of soma-produced proteins for cell growth and maintenance given there are minimal or no ribosomes in axons to synthesize proteins. Neurons are highly polarized cells with long axons extending out 1000 times the length of their cell bodies 1, 2. 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. A 10☌ decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36–14☌. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. 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. Microfluidic devices guide the growth of neurons and allow separately-controlled microenvironment for cell bodies or axon termini.

Here, we employed a microfluidic culture platform to achieve background reduction for single molecule imaging in live neurons. Despite being an increasingly popular technique, single molecule imaging in live cells is often limited by background fluorescence.

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.

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.

Mulab 7 free activation