Adapting Image J to Bone Adapting Image J to Bone HistomorphometryHistomorphometry

Kevin Egan 3.29.08Kevin Egan 3.29.08

““Practitioners of Bone Histomorphometry Practitioners of Bone Histomorphometry communicate with each other in a variety communicate with each other in a variety of arcane languages, which in general are of arcane languages, which in general are unintelligible to those outside the field.”unintelligible to those outside the field.”

Parfitt et al. "Parfitt et al. "Journal of Bone and Mineral ResearchJournal of Bone and Mineral Research 2(1987): 595-610. 2(1987): 595-610.

BackgroundBackground

Can be performed with either advanced Can be performed with either advanced imaging (microCT) or stained tissue imaging (microCT) or stained tissue sectionssections– MicroCT provides more accurate MicroCT provides more accurate

measurement of the 3D structures. measurement of the 3D structures. – But is limited to measuring mineralized But is limited to measuring mineralized

surfaces and is expensive.surfaces and is expensive.– Stained sections allow for measuring of Stained sections allow for measuring of

actively mineralizing surfaces.actively mineralizing surfaces.

BackgroundBackground

Quantitative analysis of stained tissue Quantitative analysis of stained tissue sections. Digital images of sections are sections. Digital images of sections are taken and software measures basic taken and software measures basic defined parameters within the sampledefined parameters within the sample

Commercially available solutions require Commercially available solutions require purchasing entire computer setups in purchasing entire computer setups in additions to expensive proprietary additions to expensive proprietary softwaresoftware

BackgroundBackground

With a normal microscope With a normal microscope and camera setup, and camera setup, researchers should be able researchers should be able to perform calculations to perform calculations without expensive software without expensive software suitessuitesUsing Photoshop to Using Photoshop to automatically select the automatically select the bone areas from an H/E bone areas from an H/E stain, we can use Image J to stain, we can use Image J to measure the bone within the measure the bone within the sample.sample.

Image JImage J

Image J counts the number of black pixels in Image J counts the number of black pixels in the image. From these, it derives basic the image. From these, it derives basic measurements.measurements.

Before working with complex samples, we Before working with complex samples, we need to make sure it accurately calculates need to make sure it accurately calculates these measurementsthese measurements– Distance between pointsDistance between points– AreaArea– Length (perimeter)Length (perimeter)– NumberNumber

TestingTesting

Create basic objects with Create basic objects with easy to calculate dimensions. easy to calculate dimensions. – 2 * 2 inch square. 2 * 2 inch square.

Area: 4 inchesArea: 4 inches– Perimeter: 8 inchesPerimeter: 8 inches

Create increasingly complex Create increasingly complex objects inside and see how objects inside and see how they affect the calculationsthey affect the calculations

Box in a Box test

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Are

a (i

nch

es)

Small inside box

White middle box

Black outside box

Small inside box 0.25

White middle box 0.75 0 0.75

Black outside box 3 4 3.25

Box test separated

ControlBox test

combined

βox within a box test

Circle in a Circle test

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Are

a of

cir

cle

(inch

es)

Inside black area

Middle white area

broad black area

Inside black area 0.719

Middle white area 1.548 1.548

broad black area 1.733 2.518

1 2

Circle within a circle test

Next stepsNext steps

Make sure Image J calculates perimeters Make sure Image J calculates perimeters correctly correctly – Error is introduced when trying to measure circular Error is introduced when trying to measure circular

objects which are composed of square pixels.objects which are composed of square pixels.– Circle with Diameter of 1 inch should have perimeter Circle with Diameter of 1 inch should have perimeter

of 3.14 (2 * pi * r = 3.14), but image J calculates the of 3.14 (2 * pi * r = 3.14), but image J calculates the perimeter to be 3.3perimeter to be 3.3

Will search for plugins which utilize different Will search for plugins which utilize different algorithms for calculating circular objects.algorithms for calculating circular objects.