IEEE Trans Image Process - 3-D reconstruction of microtubules from multi-angle total internal reflection fluorescence microscopy using Bayesian framework.

Tópicos

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Resumo

Total internal reflection fluorescence (TIRF) microscopy excites a thin evanescent field which theoretically decays exponentially. Each TIRF image is actually the projection of a 3-D volume and hence cannot alone produce an accurate localization of structures in the z-dimension, however, it provides 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 reconstruct the 3-D volume of the sample. With the ultimate goal to quantify important biological parameters of microtubules, we present a method to reconstruct 3-D position and orientation of microtubules based on multi-angle TIRF data, as well as experimental calibration of the actual decay function of the evanescent field at each angle. We validate our method using computer simulations, by creating a phantom simulating the curvilinear characteristics of microtubules and project the artificially constructed volume into a set of TIRF image for different penetration depth. The reconstructed depth information for the phantom data is shown to be accurate and robust to noise. We apply our method to microtubule TIRF images of PtK(2) cells in vivo. By comparing microtubule curvatures of the reconstruction results and several electron microscopy (EM) images of vertically sliced sample of microtubules, we find that the curvature statistics of our reconstruction agree well with the ground truth (EM data). Quantifying the distribution of microtubule curvature reveals an interesting discovery that microtubules can buckle and form local bendings of considerably small radius of curvature which is also visually spotted on the EM images, while microtubule bendings on a larger scale generally have a much larger radius and cannot bear the stress of a large curvature. The presented method has the potential to provide a reliable tool for 3-D reconstruction and tracking of microtubules.

Resumo Limpo

total intern reflect fluoresc tirf microscopi excit thin evanesc field theoret decay exponenti tirf imag actual project d volum henc alon produc accur local structur zdimens howev provid great improv axial resolut biolog sampl multipl angletirf microscopi allow control variat incid angl illumin laser beam thus generat set imag differ penetr depth potenti reconstruct d volum sampl ultim goal quantifi import biolog paramet microtubul present method reconstruct d posit orient microtubul base multiangl tirf data well experiment calibr actual decay function evanesc field angl valid method use comput simul creat phantom simul curvilinear characterist microtubul project artifici construct volum set tirf imag differ penetr depth reconstruct depth inform phantom data shown accur robust nois appli method microtubul tirf imag ptk cell vivo compar microtubul curvatur reconstruct result sever electron microscopi em imag vertic slice sampl microtubul find curvatur statist reconstruct agre well ground truth em data quantifi distribut microtubul curvatur reveal interest discoveri microtubul can buckl form local bend consider small radius curvatur also visual spot em imag microtubul bend larger scale general much larger radius bear stress larg curvatur present method potenti provid reliabl tool d reconstruct track microtubul

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