Single-Molecule Localization Microscopy in vivo at the Drosophila Neuromuscular Junction

Area of Science:

• Cellular and Molecular Biophysics
• Microscopy and Imaging Techniques
• Neurobiology

Background:

• Single-molecule localization microscopy (SMLM) offers nanoscale insights into cellular functions.
• Current real-time SMLM primarily uses in vitro/in cellulo models, limiting in vivo applicability.
• Understanding molecular dynamics in complex organisms requires advanced imaging methods.

Purpose of the Study:

• To provide a guide for researchers transitioning to in vivo or ex vivo SMLM.
• To highlight challenges in studying nanoscale cellular environments within multicellular organisms.
• To present a practical protocol for in vivo SMLM in a relevant biological system.

Main Methods:

• Development and description of a protocol for in vivo SMLM.
• Application of the protocol at the transgenic Drosophila melanogaster larval neuromuscular junction.
• Focus on overcoming technical obstacles for live imaging in complex systems.

Main Results:

• Successful implementation of in vivo SMLM at the Drosophila larval neuromuscular junction.
• Demonstration of direct visualization of molecules in synaptic communication.
• Identification of key challenges and strategies for in vivo SMLM.

Conclusions:

• The described protocol enables visualization of synaptic molecules in vivo.
• The methodology can be adapted for other live multicellular organisms and systems.
• This work facilitates the study of molecular dynamics in their native biological context.