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

bybyKimerly A. Powell, Ph.D.Kimerly A. Powell, Ph.D.

The Cleveland Clinic FoundationThe Cleveland Clinic Foundation

Quantitative MicroscopyQuantitative Microscopy and Micro-CTand Micro-CT


Quantitative microscopyQuantitative microscopy


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.


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


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


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


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)


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.


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


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


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


Small Animal ImagingSmall Animal Imaging


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


Fracture Model – 3D ReconstructionsFracture Model – 3D Reconstructions

Left legLeft leg Right legRight leg

A B C

osteotomiesosteotomies


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


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


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


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


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


Trabecular Architecture – Trabecular Architecture – In vivoIn vivo imaging imaging

Trabecular morphometryTrabecular morphometry

Treatment therapies - PtHTreatment therapies - PtH

Proximal tibia ratProximal tibia rat


Mouse PhenotypingMouse Phenotyping


Mouse PhenotypingMouse Phenotyping

E18.5 WT mouseE18.5 WT mouse


BA C

Application – Embryonic DevelopmentApplication – Embryonic Development

E18.5 in ethanolE18.5 in ethanol Alizarin red/Alcian BlueAlizarin red/Alcian Blue after stainingafter staining


E12.5 E13.5 E14.5

E15.5 E16.5 E17.5



E12.5 E14.5

E15.5 E16.5 E17.5

E13.5



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


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


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



Procrustes Analysis:Procrustes Analysis:

• 3D Landmark Data3D Landmark Data

• Separates Size from ShapeSeparates Size from Shape

3D reconstruction rat skull3D reconstruction rat skull


Foramen MagnumForamen Magnum Inner EarInner Ear Teeth and palletteTeeth and pallette

S

IIMM



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


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


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)


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


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


Application – Mouse HistologyApplication – Mouse Histology


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

Documents

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