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

Quantitative microscopy

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.

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

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

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

Colony Statistics for Plating Density of 2 x 106 (n=12 chambers) Mouse Bone Stem Cells Colony Statistics for Plating Density of 2 x 106 (n=12 chambers) WT KO Number Colonies 168 (35) 82 (37) Number Cells/colony 71 (102) 56 (133) Size Colony (mm2) 0.214 (0.343) 0.161 (0.343) Cell Density (%) 15.6 (6.7) 17.0 (8.5) AP- Pos Staining (%) 32.7 (23.7) 22.8 (24.1)

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.

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

CCF Micro-CT Imaging System X-ray Source Rotational Stage Image Intensifier & CCD camera 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

Micro-CT Imaging Applications Bone trabecular morphometry iliac crest, calcaneous, femoral head Tissue engineering Bone density and location of new bone formation in 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

Small Animal Imaging

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

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

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

Fracture Model - Morphometrics

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

Fracture Model - Results Table 1. Normalized Bone Volume Weeks Post-Surgery Number Samples Mean Standard Deviation 18 1.00 0.00 1 1.13 0.04 2 1.22 0.08 3 16 1.21 0.09 4 1.20 0.10 5 14 1.24 0.07

Fracture Model - Results Table 2. Normalized Callus Volume Weeks Post-Surgery Number Samples Mean Standard Deviation 18 0.00 1 0.16 0.08 2 0.49 0.18 3 16 0.23 4 0.42 5 14 0.41 0.04

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

Mouse Phenotyping

Mouse Phenotyping E18.5 WT mouse

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

Application – Embryonic Development

Application – Embryonic Development

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

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

Inner canthal Distance Application – Skull Morphometry WT MMP14-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) 4.9 mm Lower Jaw (right) 7.3 4.9

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

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

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

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

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)

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

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

Application – Mouse Histology

Acknowledgements Collaborators: 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) NIH DOD