Strategic and practical guidelines for successful structured illumination microscopy

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d

An Investigation Of Vesicular Trafficking And Secretion In Type Iii Neuromuscular Junctions Of D. Melanogaster

Bursicon is a neurohormone packaged in and secreted by type III synaptic contacts. In comparison to the depth of information describing type I and type II neuromuscular junctions (NMJs), type III NMJs are notably ill-defined. As such, this lab has provided evidence identifying bursicon as a reliable marker in an aim to characterize type III NMJs. Firstly, a comprehensive registry of proteins defining type III NMJs can be compiled through antibody-labeled colocalizations using bursicon as the type III marker. Candidates to be stained with bursicon include established presynaptic markers such as the SNARE proteins. Positional overlap in these dual antibody-staining profiles will be confirmed by sequential-excitation confocal microscopy. This proteomic approach establishes an immunohistological staining profile characterizing type III NMJs. Secondly, this effort in effect also produces a directory identifying proteins involved in vesicle delivery in type III NMJs and can be used to reveal the machinery by which neuropeptides are trafficked and secreted. While what is known about the mechanisms of vesicle trafficking employed by neurotransmitter-releasing type I synapses continues to be expanded, information on these same properties in peptidergic type III NMJs idles. Therefore, using a GFP- v variant fusion protein able to mimic bursicon's packaging and transport in vesicles, this lab has perturbed in vivo delivery of a fluorescent neuropeptide to describe type III synaptic release. In doing so we aim to elucidate the dynamic process specifically employed by type III NMJs when trafficking secretory vesicles through boutons in parallel and add to the breadth of knowledge gathered about the mechanism used during peptide release. v