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by Kimerly A. Powell, Ph.D.

The Cleveland Clinic Foundation. Quantitative Microscopy and Micro-CT. by Kimerly A. Powell, Ph.D. Quantitative microscopy. Objective.

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by Kimerly A. Powell, Ph.D.

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  1. The Cleveland Clinic Foundation Quantitative Microscopy and Micro-CT by Kimerly A. Powell, Ph.D.

  2. Quantitative microscopy

  3. Objective To determine the quantitative morphometrics (i.e., number, size, orientation) of biological structures (i.e., cells, cell nuclei, collagen fibers) in an automated unbiased fashion.

  4. Feature Analysis – Rat Tendon Collagen Fibrils Number fibrils = 247 Mean (s.d.) Area = 16701.32 (791.6) nm Range Area = [857.7 - 48203.3] nm Mean (s.d.) Diameter = 134.2 (57.0) nm Range Diameter = [33.0 - 247.7] nm

  5. Mouse Alveolar Septae Width MAT overlayed EDM Histogram EDM image mean half-width = 8.3 (0.03) pixels mean width = 11.5 microns

  6. Ncells = 189, 1.02 mm2, 10.2% density Ncolonies = 10 Mouse Bone Stem Cells – Colony Proliferation

  7. Mouse Bone Stem Cells

  8. Introduction - Micro-CT Imaging Micro-CT is a high resolution version of X-ray tomographic imaging (resolution = 10 - 100 microns). It has primarily been used to image ex vivo bone core specimens. And more recently been used to image various bone structures in vivo in small animal models.

  9. Introduction –Micro-CT Imaging X-ray source Detector Object rotates • increased scanning speed • more efficient use of x-rays • reduced dose Cone-Beam Micro-CT

  10. Image Intensifier & CCD camera X-ray Source Rotational Stage CCF Micro-CT Imaging System • 4 micron spot size X-ray • 10-225 kV, 0.01 - 0.3 mA • 3-field II (5, 7, 9 inch FOV) • 2k x 2k 12-bit CCD camera • 7 - axis positioning system

  11. Micro-CT Imaging Applications • Bone trabecular morphometry • iliac crest, calcaneous, femoral head • Tissue engineering • Bone densityand location of new bone formationin HA and PCL bone tissue scaffolds • In-vivo small animal imaging • Longitudinal evaluation of callus and bone volume in in vivo bone fracture/healing model • Mouse Phenotyping • Morphometric analysis of skeletal structure in metalloprotease knockout mice

  12. Small Animal Imaging

  13. detector x-ray source Fracture Model - Image Acquisition resolution = 100 mm Projection radiograph Hind limbs secured in micro-CT system

  14. A B C osteotomies Fracture Model – 3D Reconstructions Right leg Left leg

  15. Registered Unregistered Fracture Model – Spatial Registration • Segment bone – global threshold • Find outer outlines • ICP registration • reference – prinicpal axis week 0 ROI Red = week 0 Yellow = week 5

  16. Fracture Model - Morphometrics

  17. F E B C D A Week 1 Week 0 Week 2 Week 5 Week 3 Week 4 Fracture Model - Morphometrics

  18. Fracture Model - Results

  19. Fracture Model - Results

  20. Trabecular Architecture – In vivo imaging Trabecular morphometry Treatment therapies - PtH Proximal tibia rat

  21. Mouse Phenotyping

  22. Mouse Phenotyping E18.5 WT mouse

  23. A B C Application – Embryonic Development Alizarin red/Alcian Blue after staining E18.5 in ethanol

  24. E12.5 E13.5 E14.5 E15.5 E16.5 E17.5 Application – Embryonic Development

  25. E12.5 E13.5 E14.5 E15.5 E16.5 E17.5 Application – Embryonic Development

  26. Soft Tissue Micro-CT Imaging – Rat Embryo (E18) cut plane heart liver intestines Side view Front view

  27. Ex vivo Micro-CT Imaging of Mouse Skulls 2 week old Wild Type 2 week old MMP-14 KO

  28. Application – Skull Morphometry

  29. Application – Skull Morphometry Procrustes Analysis: • 3D Landmark Data • Separates Size from Shape 3D reconstruction rat skull

  30. Inner Ear Foramen Magnum Teeth and pallette Application – Skull Morphometry M I S

  31. Application – Vertebral Morphometry • Segment bone • Label separate objects • Separate ‘touching’ objects • Make linear and volumetric measurements on separated objects S1 S2 S3 Sacral spine of 2-week old WT mouse

  32. Application – Vertebral Morphometry S2 S2 Spinous process verterbal foramen articular process transverse process 2 week old MMP-14 KO 2 week old Wild Type

  33. Application – Vertebral Morphometry – S2

  34. secondary center of ossification Application – Tibia Morphometry growth plate trabeculae Internal micro-architecture of 2 week old mouse tibia resolution = 15 microns

  35. Application – Skeletal Atlas • Automatically label individual bones in skeleton • Standardize measurements for individual bones

  36. Application – Mouse Histology

  37. Acknowledgements Collaborators: Suneel Apte, MBBS D. Phil. Ron Midura, Ph.D. George Muschler, M.D. Don Stredney (OSC) Barry Kuban, B.S. Larry Latson, M.S. Craig Bennetts B.S. BME Prototype Lab Jason Bryan (OSC) NIH DOD

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