A correlation-based tool for quantifying membrane periodic skeleton associated periodicity.

INTRODUCTION: The advent of super-resolution microscopy revealed the membrane-associated periodic skeleton (MPS), a specialized neuronal cytoskeletal structure composed of actin rings spaced 190 nm apart by two spectrin dimers. While numerous ion channels, cell adhesion molecules, and signaling proteins have been shown to associate with the MPS, tools for accurate and unbiased quantification of their periodic localization remain scarce. METHODS: We developed Napari-WaveBreaker (https://github.com/SamKVs/napari-k2-WaveBreaker), an open-source plugin for the Napari image viewer. The tool quantifies MPS periodicity using autocorrelation and assesses periodic co-distribution between targets using cross-correlation. Performance was evaluated using both simulated datasets and STED microscopy images of periodic and non-periodic axonal proteins. RESULTS: Napari-WaveBreaker output parameters accurately reflected the visually observed periodicity and detected spatial shifts between two periodic targets. The approach was robust across varying image qualities and reliably distinguished periodic from non-periodic protein distributions. DISCUSSION: Napari-WaveBreaker provides an unbiased, quantitative framework for analyzing MPS-associated periodicity and co-distribution enabling new insights into the molecular organization and modulation of the MPS.