Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "By Kimerly A. Powell, Ph.D. The Cleveland Clinic Foundation Quantitative Microscopy and Micro-CT and Micro-CT."— Presentation transcript:

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

2 The Cleveland Clinic Foundation Quantitative microscopy

3 The Cleveland Clinic Foundation 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 The Cleveland Clinic Foundation Feature Analysis – Rat Tendon Collagen Fibrils Number fibrils = 247 Mean (s.d.) Area = (791.6) nm Range Area = [ ] nm Mean (s.d.) Diameter = (57.0) nm Range Diameter = [ ] nm

5 The Cleveland Clinic Foundation Mouse Alveolar Septae Width mean half-width = 8.3 (0.03) pixels mean width = 11.5 microns EDMimage MAT overlayed EDM Histogram

6 The Cleveland Clinic Foundation Mouse Bone Stem Cells – Colony Proliferation N cells = 189, 1.02 mm 2, 10.2% density N colonies = 10

7 The Cleveland Clinic Foundation Mouse Bone Stem Cells Colony Statistics for Plating Density of 2 x 10 6 (n=12 chambers) WTKO Number Colonies 168 (35) 82 (37) Number Cells/colony 71 (102) 56 (133) Size Colony (mm 2 ) (0.343) (0.343) Cell Density (%) 15.6 (6.7) 17.0 (8.5) AP- Pos Staining (%) 32.7 (23.7) 22.8 (24.1)

8 The Cleveland Clinic Foundation Introduction - Micro-CT Imaging Micro-CT is a high resolution version of X- ray tomographic imaging (resolution = 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 The Cleveland Clinic Foundation Introduction –Micro-CT Imaging increased scanning speed increased scanning speed more efficient use of x-rays more efficient use of x-rays reduced dose reduced dose Cone-Beam Micro-CT Detector X-raysource Objectrotates

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

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

12 The Cleveland Clinic Foundation Small Animal Imaging

13 The Cleveland Clinic Foundation Fracture Model - Image Acquisition Hind limbs secured in micro-CT system Projection radiograph x-ray source detector resolution = 100 m

14 The Cleveland Clinic Foundation Fracture Model – 3D Reconstructions Left leg Right leg ABCosteotomies

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

16 The Cleveland Clinic Foundation Fracture Model - Morphometrics

17 The Cleveland Clinic Foundation Fracture Model - Morphometrics A B CD E F Week 0 Week 1 Week 2 Week 3 Week 4 Week 5

18 The Cleveland Clinic Foundation Fracture Model - Results Table 1. Normalized Bone Volume Weeks Post- Surgery Number Samples Mean Standard Deviation

19 The Cleveland Clinic Foundation Fracture Model - Results Table 2. Normalized Callus Volume Weeks Post- Surgery Number Samples Mean Standard Deviation

20 The Cleveland Clinic Foundation Trabecular Architecture – In vivo imaging Trabecular morphometry Treatment therapies - PtH Proximal tibia rat

21 The Cleveland Clinic Foundation Mouse Phenotyping

22 The Cleveland Clinic Foundation Mouse Phenotyping E18.5 WT mouse

23 The Cleveland Clinic Foundation BAC Application – Embryonic Development E18.5 in ethanol Alizarin red/Alcian Blue after staining

24 The Cleveland Clinic Foundation E12.5E13.5E14.5 E15.5 E16.5E17.5 Application – Embryonic Development

25 The Cleveland Clinic Foundation E12.5E14.5 E15.5E16.5E17.5 E13.5 Application – Embryonic Development

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

27 The Cleveland Clinic Foundation Ex vivo Micro-CT Imaging of Mouse Skulls 2 week old Wild Type 2 week old MMP-14 KO

28 The Cleveland Clinic Foundation Application – Skull Morphometry WTMMP14-KO Skull Length 15.6 mm 11.5 mm Skull Width 8.5 mm 7.3 mm Nose Length 10.2 mm 7.4 mm Inner canthal Distance 4.5 mm 3.8 mm Upper Jaw (left) 9.7 mm 6.6 mm Upper Jaw (right) 9.8 mm 6.8 mm Lower Jaw (left) 7.3 mm 4.9 mm Lower Jaw (right)

29 The Cleveland Clinic Foundation Application – Skull Morphometry Procrustes Analysis: 3D Landmark Data 3D Landmark Data Separates Size from Shape Separates Size from Shape 3D reconstruction rat skull

30 The Cleveland Clinic Foundation Foramen Magnum Inner Ear Teeth and pallette S I M Application – Skull Morphometry

31 The Cleveland Clinic Foundation Application – Vertebral Morphometry Sacral spine of 2-week old WT mouse Segment boneSegment bone Label separate objectsLabel separate objects Separate touching objectsSeparate touching objects Make linear and volumetricMake linear and volumetric measurements on separated objects S1 S2 S3

32 The Cleveland Clinic Foundation Application – Vertebral Morphometry 2 week old Wild Type 2 week old MMP-14 KO S2 S2 Spinous process articularprocess transverseprocess verterbalforamen

33 The Cleveland Clinic Foundation Application – Vertebral Morphometry – S2 WT (n=4) MMP14-KO (n=4) Transverse Process 3.95 (0.11) 4.00 (0.38) Articular Process 1.54 (0.17) 2.51 (0.27) Spinous Process 2.11 (0.06) - Foramen Height 1.10 (0.03) 1.25 (0.09) Foramen Width 1.27 (0.16) 2.03 (0.13) Body Height 1.11 (0.02) 0.84 (0.08) Body Width 1.38 (0.06) 1.66 (0.14) Body Depth 2.07 (0.05) 1.49 (0.16)

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

35 The Cleveland Clinic Foundation Application – Skeletal Atlas Automatically label individual bones in skeleton Automatically label individual bones in skeleton Standardize measurements for individual bones Standardize measurements for individual bones

36 The Cleveland Clinic Foundation Application – Mouse Histology

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


Download ppt "By Kimerly A. Powell, Ph.D. The Cleveland Clinic Foundation Quantitative Microscopy and Micro-CT and Micro-CT."

Similar presentations


Ads by Google