Quantitative Characterization of Microtubule Ultrastructure Based on Single-Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) enables visualization of cytoskeletal architecture at nanoscale, uncovering ultrastructural details obscured in conventional imaging. In this study, we present a quantitative framework for characterizing microtubule continuity and integrity based on SMLM super-resolution imaging. We first applied this approach to evaluate the effects of various chemical fixation protocols on microtubule structural preservation. While conventional immunofluorescence imaging suggested intact microtubules after paraformaldehyde (PFA) fixation, SMLM revealed substantial fragmentation. To address this, we developed a computational algorithm that quantifies microtubule fragmentation using a defined fragmentation index (FI). Under identical 30-min fixation, quantitative analysis revealed a fragmentation hierarchy: 4% PFA > methanol > 1% glutaraldehyde (GA) ≈ 3% PFA + 0.1% GA, with the PFA-GA combination offering superior structural integrity and minimal background noise. Although prolonged PFA fixation improved preservation, it remained inferior to PFA-GA co-fixation. Notably, even a 10-min PFA-GA treatment was sufficient for effective stabilization. We further applied our framework to quantify microtubule length index (LI) in nocodazole-treated cells, revealing a drug-specific, dose-dependent microtubule disassembly. Together, we develop a quantitative pipeline based on SMLM, which establishes PFA-GA co-fixation as an optimal protocol for microtubule imaging and provides a scalable tool for super-resolution-based pharmacological screening.