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In this study, we present a quantitative framework for characterizing microtubule continuity and integrity based on SMLM super\u2010resolution 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\u2010min fixation, quantitative analysis revealed a fragmentation hierarchy: 4% PFA\u2009&gt;\u2009methanol &gt;\u20091% glutaraldehyde (GA)\u2009\u2248\u20093% PFA\u2009+\u20090.1% GA, with the PFA\u2010GA combination offering superior structural integrity and minimal background noise. Although prolonged PFA fixation improved preservation, it remained inferior to PFA\u2010GA co\u2010fixation. Notably, even a 10\u2010min PFA\u2010GA treatment was sufficient for effective stabilization. We further applied our framework to quantify microtubule length index (LI) in nocodazole\u2010treated cells, revealing a drug\u2010specific, dose\u2010dependent microtubule disassembly. Together, we develop a quantitative pipeline based on SMLM, which establishes PFA\u2010GA co\u2010fixation as an optimal protocol for microtubule imaging and provides a scalable tool for super\u2010resolution\u2010based pharmacological screening.<\/jats:p>","DOI":"10.1002\/cm.70017","type":"journal-article","created":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T09:35:37Z","timestamp":1753868137000},"page":"547-556","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Quantitative Characterization of Microtubule Ultrastructure Based on Single\u2010Molecule Localization Microscopy"],"prefix":"10.1002","volume":"83","author":[{"given":"Zhao","family":"Xie","sequence":"first","affiliation":[{"name":"The Key Laboratory of Weak\u2010Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics Nankai University  Tianjin 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