The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid with some molecules returning to the plasma membrane with a <5 minutes. Existing methods to study these trafficking pathways utilize chemical, radioactive, or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay, based on a newly designed cell-impermeable, fluorogenic ligand for HaloTag: 'Janelia Fluor 635i' (JFi; i=impermeant) which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

The rapid advancement of live-cell imaging technologies has enabled biologists to generate high-dimensional data to follow biological movement at the microscopic level. Yet, the "perceived" ease of use of modern microscopes has led to challenges whereby sub-optimal data are commonly generated that cannot support quantitative tracking and analysis as a result of various ill-advised decisions made during image acquisition. Even optimally acquired images often require further optimization through digital processing before they can be analyzed. In writing this article, we presume our target audience to be biologists with a foundational understanding of digital image acquisition and processing, who are seeking to understand the essential steps for particle/object tracking experiments. It is with this targeted readership in mind that we review the basic principles of image-processing techniques as well as analysis strategies commonly used for tracking experiments. We conclude this technical survey with a discussion of how movement behavior can be mathematically modeled and described. © 2019 by John Wiley & Sons, Inc.

To smooth the academic-to-industry transition, one institution is experimenting with offering biomedical researchers pre-commercial open access to new optical imaging systems still under development. The approach, the authors of this case study suggest, can be a win on both sides.