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Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection Fluorescence Microscopy (TIRFM) PowerPoint Presentation
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Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection Fluorescence Microscopy (TIRFM)

Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection Fluorescence Microscopy (TIRFM)

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Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection Fluorescence Microscopy (TIRFM)

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  1. Segmentation and 3D Reconstruction of Microtubules in Total Internal Reflection Fluorescence Microscopy (TIRFM) Stathis Hadjidemetriou, Derek Toomre, James S. Duncan Yale University, School of Medicine, New Haven, CT 06520

  2. Significance of Microtubules • Polymers of cytoplasm,width≈25nm • Cell structure • Cell physiology: • Dynamic, polymerize • Transport tracks • Regulate cell migration • Regulate cell division

  3. Importance in Cell Pathology • Cancer: • Growth & metastasis • Anticancer drugs target MTs: Taxol®, Vinblastine • Neurodegenerative diseases: Alzheimer’s Interaction of MTs with Cell Membrane • Regulate cell adhesions and cell migration: • Steer growth cones of neural axis • Cancer metastasis

  4. Total Internal Reflection Fluorescence Microscopy • Fluorescent tubulin • TIRF microscopy: Low photobleaching, exponential falloff with z z Cytosol Glass Iz dp Evanescent wave, dp=100 nm Microtubules dp-penetration depth ncytosol=1.33 nglass=1.52 λlaser=445nm ωTIR=61o ω Laser

  5. TIRF Microscopy Data and Processing • Algorithm: • Segmentation of MTs in 2D • Reconstruction of MTs in 3D • Evaluate MT extraction • Quantify interaction of MTs with cell membrane

  6. Relevant Work in Tubular Segmentation • Cellular biopolymers: • Actin [Work et al, 92], MTs [Jiang et al, 04, Hadjidemetriou et al, 04] • Chromosomes [Noordmans et al, 98] • Tubular organs: • Vasculature [Chung et al, 01], catheter[Weichert et al, 03] • WM fibers [Parker et al, 02],colon [Cohen et al, 01]

  7. Microtubule end, pend . Microtubule • Algorithm: • Pixel costs in neighborhood: • C1:Anisotropic for MT centerline neighborhood . x Microtubule segment Microtubule Extraction in 2D • Start from outer (+) end pend • Extract consecutive microtubule segments, S(s)

  8. Accumulate pixel costs from border to get U0(x,y): • Streamline of U0(x,y) from S(0)=prec gives S(s): . 1 C1(x) . Imax I(x) MT Extraction in 2D (cont’d) • C2: Isotropic favors microtubule • fluorescence:

  9. Curve segment S(s) w w w Inner zone Outer zones . . . prec Uo S(s) MT Segment Extraction and Evaluation MTtangent Registered MT Recently segmented point . S(s) 4w Ulo=0

  10. Microtubule End Tangent in 3D Compute depth (z) of points close to MT end to form a 3D cloud MT tangent Use point cloud to compute first eigenvector that gives the MT tangent z y . . . . . . . . . . . . . . . . φend MT x&y- image plane x φend – elevation in z of MT end

  11. Experiments with Phantoms • Phantom for noise: SNR=8.3, • Phantom for curvature: λMT=65, λMT -MT wavelength on x-y

  12. Experiments with Phantoms • Proximity: δy=6,δy≥w, w-MT width • Intersection: δθ=28o, θ-MT azimuth on x-y Size=150x150 tavg=29 sec

  13. Experiment with Real Data Fibroblast & epithelial cells • Size=179x150 • Δt=1 sec • Resolution=135 nm/pixel • t=3 min 16 sec • Error=1.9 pixels • zend: μ=54 nm, σ=31 nm • zend - depth on z of MT end • φend: μ=0.70o,σ=0.90o • φend -elevation on z of MT end Form long and close contacts useful for cell repositioning

  14. Experiment with Real Data • Size=484x632 • t= 40 min 56 sec • Error=1.8 pixels • zend: μ= 72.0 nm, • σ= 34.1 nm • φend: μ=1.2o, • σ=1.5o

  15. Experiment with Real Data • Size=421x381 • t=26 min • Error=1.6 pixels • zend: μ=104.0 nm, • σ=30.1 nm • φend:μ=1.0o, • σ=1.8o Form long and close contacts useful for cell repositioning

  16. Summary and Discussion • Image microtubules next to cell membrane • Algorithm: • Segmentation of MTs in 2D • Reconstruction of MTs in 3D • Examine interaction of MTs with cell membrane significant for cell adhesion and migration • Next: MT biomechanics such as rigidity and force