Multipolarization dark-field imaging of single endosomes in microfluidic neuronal culture for simultaneous orientation and displacement tracking

Abstract

Robust axonal transport of endosomes is critical for neuronal function. Increasingly it is appreciated that single axonal endosomes are transported by teams of molecular motors. How these motors are regulated remains poorly understood. In part, this is due to the high variability between individual endosomes. Conventional imaging approaches also only track the position of endosomes, effectively measuring one variable in a team of several motors. Here we describe how to image transport of individual axonal endosomes using multipolarization dark-field microscopy to measure both the position and the orientation of axonal endosomes. This is accomplished in a high-throughput manner by combining the imaging approach with microfluidic cell culture.