X10 expansion microscopy optimization for the lateral superior olive nucleus in the auditory brainstem

Abstract

The diffraction limit of light hinders our ability to study the interactions between biomolecules. We are limited by conventional light microscopes to a lateral resolution of approximately 200 nm. Ways exist to image at nanometer scale resolution, such as with the use of electron microscopes, but electron microscopy is not appropriate for all questions. Innovations in light microscopy over the last few decades have created a new field of imaging, known as super-resolution microscopy, where both fixed and living tissue can be imaged using multiple markers past the resolution limit of light. An array of super-resolution techniques exist, each with its own advantages and disadvantages. Here, I set out to optimize the X10 expansion microscopy technique for use in the brainstem. Located in the auditory brainstem is the superior olivary complex, where an intricate refinement process of inhibitory connections occurs between the surrounding auditory nuclei. Of interest is the inhibitory projection from the medial nucleus of the trapezoid body to the lateral superior olive, which releases the excitatory neurotransmitter glutamate during an early developmental period. For my Master of Science research project, I have worked on optimizing the X10 expansion microscopy protocol for the auditory brainstem, with a focus on the lateral superior olive nucleus. After optimization, I was able to achieve an expansion factor close to 10 in both adult and juvenile tissue using the X10 expansion microscopy protocol. My results exhibited no obvious abnormalities in staining in co-stained expansion microscopy experiments of vesicular neurotransmitter transporters and synaptotagmin calcium sensors in the lateral superior olive nucleus. The workflow presented here is ready for use in brainstem with secondary nanobodies or directly conjugated primary antibodies.