In vitro structural study of microtubules using quantitative fluorescence microscopy

Quantitative fluorescence microscopy is being explored as a novel approach for the study of microtubule (MT) structure, and as an accessible technique to complement traditional methods. In vivo , MTs typically consist of 13 protofilaments (pfs), but pf number varies from 9–17 when MTs are studied in vitro depending on the polymerization conditions and stabilizing agent. The current goal is to develop a procedure to quantify the number of pfs based on the average fluorescence emitted per unit length of MT, measured from digital images collected using wide‐field epifluorescence microscopy. We aim to: (1) develop a technique to quantify MT fluorescence for MTs of known pf number, and (2) quantify the fluorescence produced by MTs grown under a range of polymerization conditions, comparing putative variations in pf number. The technique may also allow for comparison between the structural properties of in vivo versus in vitro MTs, and help to explain variations in mechanical properties seen across MT types. Preliminary results indicate that our methods enable differences of as few as two pfs to be resolved, but with further improvements to the data collection methods and image analysis, this number may be improved. This work is supported by NIH Grant #1R15GM083256‐01.