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

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 = (791.6) nm
Range Area = [ ] nm
Mean (s.d.) Diameter = (57.0) nm
Range Diameter = [ ] nm

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

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

7. 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)

8. 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. 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

10. CCF Micro-CT Imaging System
X-ray
Source
Rotational
Stage
Image Intensifier & CCD camera
4 micron spot size X-ray
kV, 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 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

12. Small Animal Imaging

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

14. Fracture Model – 3D ReconstructionsB C
osteotomies
Left leg
Right leg

15. 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

16. Fracture Model - Morphometrics

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

18. 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

19. 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

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

24. Application – Embryonic Development

25. 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. 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

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

30. Application – Skull Morphometry
Foramen Magnum
Inner Ear
Teeth and pallette 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 MorphometryS2 S2
Spinous process
verterbal
foramen
articular
process
transverse
process
2 week old Wild Type
2 week old MMP-14 KO

33. 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. Application – Tibia Morphometry
secondary center of ossification
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: 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