Kimerly A. Powell, Ph.D.

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Quantitative Microscopy and Micro-CT

• by
• Kimerly A. Powell, Ph.D.

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Quantitative microscopy
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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.
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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
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Mouse Alveolar Septae Width
MAT overlayed EDM
Histogram
EDM image
mean half-width 8.3 (0.03) pixels mean width
11.5 microns
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Mouse Bone Stem Cells Colony Proliferation
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Mouse Bone Stem Cells
Colony Statistics for Plating Density of 2 x 106 (n12 chambers) Colony Statistics for Plating Density of 2 x 106 (n12 chambers) Colony Statistics for Plating Density of 2 x 106 (n12 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)
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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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Introduction Micro-CT Imaging

• increased scanning speed
• more efficient use of x-rays
• reduced dose

Cone-Beam Micro-CT
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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

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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

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Small Animal Imaging
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Fracture Model - Image Acquisition
resolution 100 mm
Projection radiograph
Hind limbs secured in micro-CT system
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Fracture Model 3D Reconstructions
Right leg
Left leg
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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
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Fracture Model - Morphometrics
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Fracture Model - Morphometrics
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Fracture Model - Results
Table 1. Normalized Bone Volume Table 1. Normalized Bone Volume Table 1. Normalized Bone Volume Table 1. Normalized Bone Volume
Weeks Post-Surgery Number Samples Mean Standard Deviation
0 18 1.00 0.00
1 18 1.13 0.04
2 18 1.22 0.08
3 16 1.21 0.09
4 16 1.20 0.10
5 14 1.24 0.07
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Fracture Model - Results
Table 2. Normalized Callus Volume Table 2. Normalized Callus Volume Table 2. Normalized Callus Volume Table 2. Normalized Callus Volume
Weeks Post-Surgery Number Samples Mean Standard Deviation
0 18 0.00 0.00
1 18 0.16 0.08
2 18 0.49 0.18
3 16 0.49 0.23
4 16 0.42 0.16
5 14 0.41 0.04
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Trabecular Architecture In vivo imaging
Trabecular morphometry Treatment therapies - PtH
Proximal tibia rat
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Mouse Phenotyping
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Mouse Phenotyping
E18.5 WT mouse
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Application Embryonic Development
E18.5 in ethanol
Alizarin red/Alcian Blue
after staining
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Application Embryonic Development
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Application Embryonic Development
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Soft Tissue Micro-CT Imaging Rat Embryo (E18)
cut plane
heart
liver
intestines
Side view
Front view
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Ex vivo Micro-CT Imaging of Mouse Skulls
2 week old Wild Type
2 week old MMP-14 KO
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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) 7.3 mm 4.9 mm
Lower Jaw (right) 7.3 4.9
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Application Skull Morphometry

• Procrustes Analysis
• 3D Landmark Data
• Separates Size from Shape

3D reconstruction rat skull
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Application Skull Morphometry
M
I
S
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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
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Application Vertebral Morphometry
S2
S2
Spinous process
verterbal foramen
articular process
transverse process
2 week old MMP-14 KO
2 week old Wild Type
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Application Vertebral Morphometry S2
WT (n4) MMP14-KO (n4)
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)
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Application Tibia Morphometry
growth plate
trabeculae
Internal micro-architecture of 2 week old mouse
tibia resolution 15 microns
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Application Skeletal Atlas

• Automatically label individual bones in skeleton
• Standardize measurements for individual bones

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Application Mouse Histology
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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