bybyKimerly A. Powell, Ph.D.Kimerly A. Powell, Ph.D.
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Quantitative MicroscopyQuantitative Microscopy and Micro-CTand Micro-CT
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Quantitative microscopyQuantitative microscopy
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ObjectiveObjective
To determine the quantitative To determine the quantitative morphometrics (morphometrics (i.e.i.e., number, size, , number, size, orientation) of biological structures (orientation) of biological structures (i.e.i.e., , cells, cell nuclei, collagen fibers) in an cells, cell nuclei, collagen fibers) in an automated unbiased fashion.automated unbiased fashion.
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Feature Analysis – Rat Tendon Collagen FibrilsFeature Analysis – Rat Tendon Collagen Fibrils
Number fibrils = 247Number fibrils = 247
Mean (s.d.) Area = Mean (s.d.) Area = 16701.32 (791.6) nm16701.32 (791.6) nm
Range Area = [857.7 - 48203.3] nmRange Area = [857.7 - 48203.3] nm
Mean (s.d.) Diameter = 134.2 (57.0) nmMean (s.d.) Diameter = 134.2 (57.0) nm
Range Diameter = [33.0 - 247.7] nmRange Diameter = [33.0 - 247.7] nm
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Mouse Alveolar Septae WidthMouse Alveolar Septae Width
Septae half-width(pixels)
0 5 10 15 20 25 30
Fre
qu
ency
0200400600800100012001400
mean half-width = 8.3 (0.03) pixelsmean half-width = 8.3 (0.03) pixelsmean width = 11.5 micronsmean width = 11.5 microns
EDMEDMimageimage
MAT overlayed MAT overlayed EDMEDM
Histogram Histogram
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Mouse Bone Stem Cells – Colony Proliferation Mouse Bone Stem Cells – Colony Proliferation
NNcellscells = 189, 1.02 mm = 189, 1.02 mm22, 10.2% density, 10.2% densityNNcolonies colonies = 10= 10
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Mouse Bone Stem CellsMouse Bone Stem Cells
Colony Statistics for Plating Density of 2 x 10Colony Statistics for Plating Density of 2 x 1066 (n=12 chambers)(n=12 chambers)
WTWT KOKO
Number ColoniesNumber Colonies 168 (35)168 (35) 82 (37)82 (37)
Number Cells/colonyNumber Cells/colony 71 (102)71 (102) 56 (133)56 (133)
Size Colony (mmSize Colony (mm22)) 0.214 (0.343)0.214 (0.343) 0.161 (0.343)0.161 (0.343)
Cell Density (%)Cell Density (%) 15.6 (6.7)15.6 (6.7) 17.0 (8.5)17.0 (8.5)
AP- Pos Staining (%)AP- Pos Staining (%) 32.7 (23.7)32.7 (23.7) 22.8 (24.1)22.8 (24.1)
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Introduction - Micro-CT ImagingIntroduction - Micro-CT Imaging
Micro-CT is a high resolution version of Micro-CT is a high resolution version of
X-ray tomographic imaging (resolution = 10 - X-ray tomographic imaging (resolution = 10 -
100 microns). It has primarily been used to 100 microns). It has primarily been used to
image image ex vivoex vivo bone core specimens. And more bone core specimens. And more
recently been used to image various bone recently been used to image various bone
structures structures in vivoin vivo in small animal models. in small animal models.
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Introduction –Micro-CT ImagingIntroduction –Micro-CT Imaging
• increased scanning speedincreased scanning speed
• more efficient use of x-raysmore efficient use of x-rays
• reduced dosereduced dose
Cone-Beam Micro-CTCone-Beam Micro-CT
DetectorDetectorX-rayX-raysourcesource
Object Object
rotatesrotates
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CCF Micro-CT Imaging SystemCCF Micro-CT Imaging System
• 4 micron spot size X-ray 4 micron spot size X-ray
• 10-225 kV, 0.01 - 0.3 mA10-225 kV, 0.01 - 0.3 mA
• 3-field II (5, 7, 9 inch FOV)3-field II (5, 7, 9 inch FOV)
• 2k x 2k 12-bit CCD camera2k x 2k 12-bit CCD camera
• 7 - axis positioning system7 - axis positioning system
X-ray
Source
Rotational
Stage
Image Intensifier & CCD camera
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Micro-CT Imaging ApplicationsMicro-CT Imaging Applications
• Bone trabecular morphometryBone trabecular morphometry• iliac crest, calcaneous, femoral headiliac crest, calcaneous, femoral head
• Tissue engineeringTissue engineering• Bone densityBone density and location of new bone formationand location of new bone formation in HA in HA and PCL bone tissue scaffoldsand PCL bone tissue scaffolds
• In-vivoIn-vivo small animal imaging small animal imaging• Longitudinal evaluation of callus and bone volume in Longitudinal evaluation of callus and bone volume in in in vivovivo bone fracture/healing model bone fracture/healing model
• Mouse PhenotypingMouse Phenotyping • Morphometric analysis of skeletal structure in Morphometric analysis of skeletal structure in metalloprotease knockout micemetalloprotease knockout mice
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Small Animal ImagingSmall Animal Imaging
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Fracture Model - Image AcquisitionFracture Model - Image Acquisition
Hind limbs secured in micro-CT systemHind limbs secured in micro-CT system Projection radiographProjection radiograph
x-ray sourcex-ray source detectordetector
resolution = 100 m
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Fracture Model – 3D ReconstructionsFracture Model – 3D Reconstructions
Left legLeft leg Right legRight leg
A B C
osteotomiesosteotomies
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Fracture Model – Spatial RegistrationFracture Model – Spatial Registration
UnregisteredUnregistered RegisteredRegistered
Red = week 0Red = week 0 Yellow = week 5Yellow = week 5
ROIROI
• Segment bone – global Segment bone – global thresholdthreshold
• Find outer outlinesFind outer outlines
• ICP registrationICP registration
• reference – prinicpal reference – prinicpal axis week 0axis week 0
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Fracture Model - MorphometricsFracture Model - Morphometrics
distance from osteotomy (mm)
-6 -4 -2 0 2 4 6
cro
ss
-se
ctio
na
l are
a (
mm
2)
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
callusbone
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Fracture Model - MorphometricsFracture Model - MorphometricsA B C D E F
Week 0Week 0 Week 1Week 1 Week 2Week 2 Week 3Week 3 Week 4Week 4 Week 5Week 5
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Fracture Model - ResultsFracture Model - ResultsTable 1. Normalized Bone VolumeTable 1. Normalized Bone Volume
Weeks Post-Weeks Post-SurgerySurgery
Number Number SamplesSamples
MeanMean Standard Standard DeviationDeviation
00 1818 1.001.00 0.000.00
11 1818 1.131.13 0.040.04
22 1818 1.221.22 0.080.08
33 1616 1.211.21 0.090.09
44 1616 1.201.20 0.100.10
55 1414 1.241.24 0.070.07
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Fracture Model - ResultsFracture Model - ResultsTable 2. Normalized Callus VolumeTable 2. Normalized Callus Volume
Weeks Post-Weeks Post-SurgerySurgery
Number Number SamplesSamples
MeanMean Standard Standard DeviationDeviation
00 1818 0.000.00 0.000.00
11 1818 0.160.16 0.080.08
22 1818 0.490.49 0.180.18
33 1616 0.490.49 0.230.23
44 1616 0.420.42 0.160.16
55 1414 0.410.41 0.040.04
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Trabecular Architecture – Trabecular Architecture – In vivoIn vivo imaging imaging
Trabecular morphometryTrabecular morphometry
Treatment therapies - PtHTreatment therapies - PtH
Proximal tibia ratProximal tibia rat
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Mouse PhenotypingMouse Phenotyping
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Mouse PhenotypingMouse Phenotyping
E18.5 WT mouseE18.5 WT mouse
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BA C
Application – Embryonic DevelopmentApplication – Embryonic Development
E18.5 in ethanolE18.5 in ethanol Alizarin red/Alcian BlueAlizarin red/Alcian Blue after stainingafter staining
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E12.5 E13.5 E14.5
E15.5 E16.5 E17.5
Application – Embryonic DevelopmentApplication – Embryonic Development
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E12.5 E14.5
E15.5 E16.5 E17.5
E13.5
Application – Embryonic DevelopmentApplication – Embryonic Development
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Soft Tissue Micro-CT Imaging – Rat Embryo (E18)Soft Tissue Micro-CT Imaging – Rat Embryo (E18)
Side viewSide view Front viewFront view
liverliver
heartheart
intestinesintestines
cut planecut plane
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Ex vivoEx vivo Micro-CT Imaging of Mouse Skulls Micro-CT Imaging of Mouse Skulls
2 week old Wild Type2 week old Wild Type 2 week old MMP-14 KO2 week old MMP-14 KO
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Application – Skull MorphometryApplication – Skull Morphometry
WTWT MMP14-KOMMP14-KO
Skull LengthSkull Length 15.6 mm15.6 mm 11.5 mm11.5 mm
Skull WidthSkull Width 8.5 mm8.5 mm 7.3 mm7.3 mm
Nose LengthNose Length 10.2 mm10.2 mm 7.4 mm7.4 mm
Inner canthal Inner canthal DistanceDistance
4.5 mm4.5 mm 3.8 mm3.8 mm
Upper Jaw (left)Upper Jaw (left) 9.7 mm9.7 mm 6.6 mm6.6 mm
Upper Jaw (right)Upper Jaw (right) 9.8 mm9.8 mm 6.8 mm6.8 mm
Lower Jaw (left)Lower Jaw (left) 7.3 mm7.3 mm 4.9 mm4.9 mm
Lower Jaw (right)Lower Jaw (right) 7.37.3 4.94.9
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Application – Skull MorphometryApplication – Skull Morphometry
Procrustes Analysis:Procrustes Analysis:
• 3D Landmark Data3D Landmark Data
• Separates Size from ShapeSeparates Size from Shape
3D reconstruction rat skull3D reconstruction rat skull
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Foramen MagnumForamen Magnum Inner EarInner Ear Teeth and palletteTeeth and pallette
S
IIMM
Application – Skull MorphometryApplication – Skull Morphometry
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Application – Vertebral MorphometryApplication – Vertebral Morphometry
Sacral spine of 2-week old WT mouseSacral spine of 2-week old WT mouse
•Segment bone Segment bone
•Label separate objectsLabel separate objects
•Separate ‘touching’ objectsSeparate ‘touching’ objects
•Make linear and volumetricMake linear and volumetric
measurements on separated measurements on separated
objectsobjects
S1S1
S2S2
S3S3
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Application – Vertebral MorphometryApplication – Vertebral Morphometry
2 week old Wild Type2 week old Wild Type 2 week old MMP-14 KO2 week old MMP-14 KO
S2S2 S2S2Spinous processSpinous process
articulararticularprocessprocess
transversetransverseprocessprocess
verterbal verterbal foramenforamen
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Application – Vertebral Morphometry – S2Application – Vertebral Morphometry – S2WT (n=4)WT (n=4) MMP14-KO (n=4)MMP14-KO (n=4)
Transverse Transverse ProcessProcess
3.95 (0.11)3.95 (0.11) 4.00 (0.38)4.00 (0.38)
Articular ProcessArticular Process 1.54 (0.17)1.54 (0.17) 2.51 (0.27)2.51 (0.27)
Spinous ProcessSpinous Process 2.11 (0.06)2.11 (0.06) --
Foramen HeightForamen Height 1.10 (0.03)1.10 (0.03) 1.25 (0.09)1.25 (0.09)
Foramen WidthForamen Width 1.27 (0.16)1.27 (0.16) 2.03 (0.13)2.03 (0.13)
Body HeightBody Height 1.11 (0.02)1.11 (0.02) 0.84 (0.08)0.84 (0.08)
Body WidthBody Width 1.38 (0.06)1.38 (0.06) 1.66 (0.14)1.66 (0.14)
Body DepthBody Depth 2.07 (0.05)2.07 (0.05) 1.49 (0.16)1.49 (0.16)
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Internal micro-architecture of 2 week old mouse tibiaInternal micro-architecture of 2 week old mouse tibia
resolution = 15 micronsresolution = 15 microns
secondary center of ossificationsecondary center of ossification
trabeculaetrabeculae
growth plategrowth plate
Application – Tibia MorphometryApplication – Tibia Morphometry
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Application – Skeletal AtlasApplication – Skeletal Atlas• Automatically label individual Automatically label individual bones in skeletonbones in skeleton
• Standardize measurements for Standardize measurements for individual bones individual bones
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Application – Mouse HistologyApplication – Mouse Histology
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AcknowledgementsAcknowledgements
Barry Kuban, B.S.Barry Kuban, B.S.
Larry Latson, M.S.Larry Latson, M.S.
Craig Bennetts B.S.Craig Bennetts B.S.
BME Prototype LabBME Prototype Lab
Jason Bryan (OSC)Jason Bryan (OSC)
Collaborators:Collaborators:
Suneel Apte, MBBS D. Phil.Suneel Apte, MBBS D. Phil.
Ron Midura, Ph.D.Ron Midura, Ph.D.
George Muschler, M.D.George Muschler, M.D.
Don Stredney (OSC)Don Stredney (OSC)
NIHNIH
DODDOD