Estimation of 3D geometry of microtubules using multi-angle total internal reflection fluorescence microscopy

Abstract

With the ultimate goal to quantify important biological parameters of microtubules, we present a method to estimate the 3D positions of microtubules from multi-angle TIRF data based on the calibrated decay profiles for each angle. Total Internal Reflection Fluorescence (TIRF) Microscopy images are actually projections of 3D volumes and hence cannot alone produce an accurate localization of structures in the z-dimension, however, they provide greatly improved axial resolution for biological samples. Multiple angle-TIRF microscopy allows controlled variation of the incident angle of the illuminating laser beam, thus generating a set of images of different penetration depths with the potential to estimate the 3D volume of the sample. Our approach incorporates prior information about intensity and geometric smoothness. We validate our method using computer simulated phantom data and test its robustness to noise. We apply our method to TIRF images of microtubules in PTK2 cells and compare the distribution of the microtubule curvatures with electron microscopy (EM) images. © 2010 Springer-Verlag.