3-D reconstruction of microtubules from multi-angle total internal reflection fluorescence microscopy using Bayesian...

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Total internal reflection fluorescence (TIRF) microscopy provides high axial resolution for biological samples but cannot alone determine 3-D structure.
• Reconstructing 3-D information from TIRF images is challenging due to the limited z-dimension localization.

Purpose of the Study:

• To develop and validate a method for reconstructing the 3-D position and orientation of microtubules using multi-angle TIRF data.
• To quantify biological parameters of microtubules, specifically their curvature and bending behavior.

Main Methods:

• Utilized multiple angle-TIRF microscopy to capture images at varying evanescent field penetration depths.
• Developed a reconstruction algorithm based on multi-angle TIRF data and experimentally calibrated evanescent field decay functions.
• Validated the method using computer simulations with a microtubule phantom and in vivo PtK(2) cell imaging.

Main Results:

• The 3-D reconstruction method accurately determined microtubule depth information, proving robust to noise in simulated data.
• Reconstructed microtubule curvature statistics from in vivo cell images closely matched electron microscopy ground truth data.
• Discovered that microtubules exhibit local, sharp bends and larger-scale, smoother curves, with limitations on maximum curvature.

Conclusions:

• The developed multi-angle TIRF method offers a reliable tool for accurate 3-D reconstruction and tracking of microtubules.
• The findings provide new quantitative insights into microtubule structural mechanics and bending behaviors within cells.
• This technique has significant potential for advancing the study of cytoskeletal dynamics and cell mechanics.