Transcript Mammography Research Overview
Quantitative Microscopy and Micro-CT
by Kimerly A. Powell, Ph.D.
The Cleveland Clinic Foundation
Quantitative microscopy
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Objective To determine morphometrics the (i.e., quantitative number, size, orientation) of biological structures (i.e., cells, cell nuclei, collagen fibers) in an automated unbiased fashion.
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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 The Cleveland Clinic Foundation
Mouse Alveolar Septae Width EDM image
1400 1200 1000 800 600 400 200 0 0 5 10 15 20 25 30
Septae half-width (pixels) MAT overlayed EDM Histogram mean half-width = 8.3 (0.03) pixels mean width = 11.5 microns The Cleveland Clinic Foundation
Mouse Bone Stem Cells – Colony Proliferation N colonies = 10 The Cleveland Clinic Foundation N cells = 189, 1.02 mm 2 , 10.2% density
Mouse Bone Stem Cells Colony Statistics for Plating Density of 2 x 10 6 (n=12 chambers) WT KO Number Colonies Number Cells/colony Size Colony (mm 2 ) Cell Density (%) AP- Pos Staining (%) 168 (35) 71 (102) 0.214 (0.343) 15.6 (6.7) 32.7 (23.7) 82 (37) 56 (133) 0.161 (0.343) 17.0 (8.5) 22.8 (24.1) The Cleveland Clinic Foundation
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.
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X-ray source Introduction –Micro-CT Imaging Detector
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increased scanning speed
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more efficient use of x-rays
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reduced dose Object rotates Cone-Beam Micro-CT The Cleveland Clinic Foundation
CCF Micro-CT Imaging System X-ray Source Rotational Stage The Cleveland Clinic Foundation Image Intensifier & CCD camera
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4 micron spot size X-ray
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10-225 kV, 0.01 - 0.3 mA
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3-field II (5, 7, 9 inch FOV)
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2k x 2k 12-bit CCD camera
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7 - axis positioning system
Micro-CT Imaging Applications
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Bone trabecular morphometry
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iliac crest, calcaneous, femoral head
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Tissue engineering
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Bone density and location of new bone formation in HA and PCL bone tissue scaffolds
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In-vivo small animal imaging
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Longitudinal evaluation of callus and bone volume in in vivo bone fracture/healing model
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Mouse Phenotyping
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Morphometric analysis of skeletal structure in metalloprotease knockout mice The Cleveland Clinic Foundation
Small Animal Imaging
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Fracture Model - Image Acquisition x-ray source detector Hind limbs secured in micro-CT system The Cleveland Clinic Foundation resolution = 100
m
m Projection radiograph
A Fracture Model – 3D Reconstructions B C osteotomies The Cleveland Clinic Foundation Left leg Right leg
Fracture Model – Spatial Registration
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Segment bone – global threshold ROI
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Find outer outlines
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ICP registration
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reference – prinicpal axis week 0 Unregistered The Cleveland Clinic Foundation Registered Red = week 0 Yellow = week 5
Fracture Model - Morphometrics
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
-6
callus bone
6 -4 -2 0 2
distance from osteotomy (mm)
4
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A Fracture Model - Morphometrics B C D E F Week 0 The Cleveland Clinic Foundation Week 1 Week 2 Week 3 Week 4 Week 5
Fracture Model - Results Table 1. Normalized Bone Volume Weeks Post Surgery 0 Number Samples 18 Mean 1.00
Standard Deviation 0.00
3 4 5 1 2 18 18 16 16 14 1.13
1.22
1.21
1.20
1.24
0.04
0.08
0.09
0.10
0.07
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Fracture Model - Results Table 2. Normalized Callus Volume Weeks Post Surgery 0 Number Samples 18 Mean 0.00
Standard Deviation 0.00
3 4 5 1 2 18 18 16 16 14 0.16
0.49
0.49
0.42
0.41
0.08
0.18
0.23
0.16
0.04
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Trabecular Architecture – In vivo imaging Trabecular morphometry Treatment therapies - PtH Proximal tibia rat The Cleveland Clinic Foundation
Mouse Phenotyping
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Mouse Phenotyping The Cleveland Clinic Foundation E18.5 WT mouse
Application – Embryonic Development
A B C
E18.5 in ethanol The Cleveland Clinic Foundation Alizarin red/Alcian Blue after staining
Application – Embryonic Development E12.5
E13.5
E14.5
E15.5
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E17.5
Application – Embryonic Development E12.5
E13.5
E14.5
E15.5
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E17.5
Soft Tissue Micro-CT Imaging – Rat Embryo (E18) cut plane Side view The Cleveland Clinic Foundation heart liver intestines Front view
Ex vivo Micro-CT Imaging of Mouse Skulls 2 week old Wild Type The Cleveland Clinic Foundation 2 week old MMP-14 KO
Application – Skull Morphometry Skull Length Skull Width Nose Length Inner canthal Distance Upper Jaw (left) Upper Jaw (right) Lower Jaw (left) Lower Jaw (right) The Cleveland Clinic Foundation WT 15.6 mm 8.5 mm 10.2 mm 4.5 mm 9.7 mm 9.8 mm 7.3 mm 7.3
MMP14-KO 11.5 mm 7.3 mm 7.4 mm 3.8 mm 6.6 mm 6.8 mm 4.9 mm 4.9
Application – Skull Morphometry Procrustes Analysis:
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3D Landmark Data
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Separates Size from Shape 3D reconstruction rat skull The Cleveland Clinic Foundation
Application – Skull Morphometry I M S Foramen Magnum Inner Ear Teeth and pallette The Cleveland Clinic Foundation
S1 S2 S3 Application – Vertebral Morphometry
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Segment bone
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Label separate objects
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Separate ‘touching’ objects
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Make linear and volumetric measurements on separated objects Sacral spine of 2-week old WT mouse The Cleveland Clinic Foundation
Application – Vertebral Morphometry S2 Spinous process articular process verterbal foramen S2 transverse process 2 week old Wild Type The Cleveland Clinic Foundation 2 week old MMP-14 KO
Application – Vertebral Morphometry – S2 WT (n=4) MMP14-KO (n=4) 3.95 (0.11) 4.00 (0.38) Transverse Process Articular Process Spinous Process Foramen Height Foramen Width Body Height Body Width Body Depth 1.54 (0.17) 2.11 (0.06) 1.10 (0.03) 1.27 (0.16) 1.11 (0.02) 1.38 (0.06) 2.07 (0.05) 2.51 (0.27) 1.25 (0.09) 2.03 (0.13) 0.84 (0.08) 1.66 (0.14) 1.49 (0.16) The Cleveland Clinic Foundation
Application – Tibia Morphometry secondary center of ossification growth plate trabeculae Internal micro-architecture of 2 week old mouse tibia The Cleveland Clinic Foundation resolution = 15 microns
Application – Skeletal Atlas
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Automatically label individual bones in skeleton
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Standardize measurements for individual bones The Cleveland Clinic Foundation
Application – Mouse Histology 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) NIH DOD The Cleveland Clinic Foundation