BIOQUANT PROTOCOLSINTRODUCTION...........................................................................................................................................3

Bioquant interface description............................................................................................................3

Create or open an existing data volume and data set.........................................................................4

Open an Image file...............................................................................................................................4

DEFINITION OF THE ROI...............................................................................................................................5

Definition of the Tissue Volume: ROI...................................................................................................5

Transposition of a pre-existing ROI......................................................................................................5

GENERAL BONE PARAMETER MEASUREMENTS: TV, BV, BV/TV, BS, BS/BV, Tb.Dm, Tb.N, Tb.Sp................6

ROI.......................................................................................................................................................6

Thresholding........................................................................................................................................6

Editing of the Threshold......................................................................................................................6

Bone parameter Measurements..........................................................................................................7

OSTEOID RELATED MEASUREMENT: OV, OV/BV, OS, OS/BS, O.Wi.............................................................8

ROI.......................................................................................................................................................8

Osteoid Thresholding/Editing..............................................................................................................8

Osteoid Measurement.........................................................................................................................9

OSTEOBLAST MEASUREMENTS: N.Ob, N.Ob/BS........................................................................................10

ROI.....................................................................................................................................................10

Osteoblast Measurements................................................................................................................10

OSTEOCLAST RELATED MEASUREMENTS: Oc.S, Oc.S/BS, N.Oc, N.Oc/BS..................................................11

ROI.....................................................................................................................................................11

Threshold for TRAP staining (osteoclasts).........................................................................................11

Refining..............................................................................................................................................11

Editing the threshold.........................................................................................................................12

Osteoclast Measurement..................................................................................................................12

FLUORESCENT LABELING MEASUREMENT: sLS, dLS, MS, MS/BS, Lr.L.Wi, MAR, BFR/BS, BFR/BV, BFR/TV, Omt...........................................................................................................................................................13

ROI.....................................................................................................................................................13

Tissue volume measurement.............................................................................................................13

Threshold for sLS...............................................................................................................................13

1

Editing the threshold for sLS..............................................................................................................13

sLS Measurement..............................................................................................................................14

dLS Measurement..............................................................................................................................14

Data Exportation.......................................................................................................................................15

2

INTRODUCTION

Bioquant interface descriptionThe Bioquant interface is divided in 5 major parts- 1 on the top the “ribbon”, which gathered all the functions for image treatment and

analysis. The “ribbon” is divided in 8 “regions”:o Guideo Imageo Selected Listo Parameterso ROI Toolso Threshold o Editingo Measurements

- 2 on the bottom left part the “Image Window”, which is used to draw the ROI, plot the objects you want to measure … etc.

- 3 on the top right, the “Large Image Navigator”. This will allow you to navigate on your section at high magnification. The area within a blue rectangle (Pan Box) will be displayed in the “Image Window”.

- 4 on the middle bottom, the “calculations” window display the data you have measured.

- 5 on the bottom right, the “Overview” window displays a map on which your measurements will appear.

3

Create or open an existing data volume and data set.A data volume is dedicated to a project or a study. It represents the place where all the data sets related to the project will be stored and associated to one another.

A data set is associated with a specific subject, animal or specimen. A new data set must be created using an existing template. A data set contains at least one and possibly many data arrays. Each array is associated with a raw data collection (common arrays include bone volume, osteoid volume, osteoblast count, etc…)

- From the File menu, choose “New Data Set” to open the “create new data set wizard”.

- Step1: Click either the “Create New” or “Use Existing” button (If you are beginning a new study, create a new data volume.) and click “next”.

- Step 2: If the “Create New” option was chosen, Type a name in the “New Data Volume Name” field i.e. “study #1” (Current date and time by default). Use the “browse” button to the desired location. The preferred location is “C:\BQDATA\your name’s data”. Click “Next”.

- Step3: Scroll through the data sets in the Data Sets list and click once to highlight the data set that is to be used as a template (Tb. Rodent – Single Slide) and click “Next”.

4

- Step4: In the New Data Set Name field, type the desired name for the data set i.e. “specimen # – section # (The default name is the current date and time.) and click “Finish”.

Open an Image file.As this station is not connected to a camera, analysis will be performed on image files stored on the computer or external drive.- In the “Image” region, select “Type: Image File”.- Then, click on “Open Single”, browse to the location where the image you want to

analyze is stored (H&E). Select it and click on the “Open” button. Your selected image will be displayed in the “Image Window” and in the “Large Image Navigator”.

- You have to set the proper magnification corresponding to your image in the “Parameters” region “Mag” field.

- In the “Parameters” region, make sure the “Topo Array” is set on “D4”.- In the “Parameters” region, make sure the “Save to Topo Array” box is checked. This

will allow you to keep track of all the measurements made on the specimen.

5

DEFINITION OF THE ROI

Definition of the Tissue Volume: ROI.This procedure will allow you to reproducibly define the boundary of the ROI 150 microns away from the growth plate and the cortical shell.

- Make sure the L3 array is selected in the “Selected List” region to make “Bone Surface” the current array.

- Set the picture size to appear in full screen view in the “Image Window” by clicking the “shrink/expand” button in the “Large Image Navigator”. (double square icon, fourth from the top in the tool bar).

- In the “ROI Tools” region select the “IROI cursor” type.- Set the Radius to 150microns.- Check the “Spacebar To End” box. - Click “Define” to begin drawing.- Click along the edges of the growth plate and the cortical walls. Tap the space bar to

close the shape.- Right click to exit the image window.

Transposition of a pre-existing ROIThe following steps allow you to transpose a pre-existing ROI.

- In the “ROI Tools”, set “type to “Topo ROI”,- Click the Define button. The redraw tracings from the TV/BV/BS section show up

(dark green).- Click once on the TV tracing to select it as the region of interest (dark green tracing

turns to light green).

6

GENERAL BONE PARAMETER MEASUREMENTS: TV, BV, BV/TV, BS, BS/BV, Tb.Dm, Tb.N, Tb.Sp

For the proper measurement of these parameters, both mineralized and unmineralized (osteoid) bones have to be taken into account. These measurements can be performed on H&E or Masson’s Trichrome stained sections.

ROIDefine or transpose the ROI as previously described

Thresholding- Make sure the L3 array is selected in the “Selected List” region to make “Bone

Surface” the current array.- To be able to see the range of colors taken into account by your threshold the

button “Show” has to be active. - Click the “Select” button in the “Threshold” region.- In the “Image Window”, click on mineralized bone. You can use the “Z” key to zoom

in. Continue to click on other regions of mineralized bone. Each click will add the selected color to the range that identifies mineralized bone. It is better to underestimate the thresholding and use the editing tools to fix it.

- To add a large region of mineralized bone, click and drag the cursor. All the selected colors will be added to the range that identifies mineralized bone.

- To undo, press the “U” key.- To exit the Image Window, right click.- If you want to start over, press the “Select” button in the “Threshold” region and

repeat.- In the Threshold region, click the Assign button to assign the threshold to the L3

array.

Editing of the Threshold.The goal of editing procedure is to clean up the outlines.- In the “Editing” region the different tools “Clean”, the “Dilate”, the “Erode” can be

used to clean up bone fragments and smooth the edges of the objects. At the end of the process you get a view of the preview outline (yellow).

- Use alternatively the “Draw” and “Erase” tools located in the “Editing” region to add osteoid, cracks and missed bone and fix any thresholding mistakes. You can use the “Z” key to zoom in.

7

Bone parameter Measurements.- In the “Threshold” region, make sure the Show button is ON so that the threshold is

visible. This gives you visual validation of measurement.- In the “Measurement” region, “Type” as to be set on “General”- In the “Measurement” region, click “Preview”, and “Measure” to record the bone

data. The threshold turns color to indicate it has been measured. Tissue Volume, Bone Volume, and Bone Surface have been measured. “Calculations” and “Overview” update.

- In the “Calculations” window, the 8 parameters will be measured or calculated are displayed on the “A” channel.

- Once the scan of the ROI is completed, Go to “File” menu and select “Save data”.

8

OSTEOID RELATED MEASUREMENT: OV, OV/BV, OS, OS/BS, O.Wi

Osteoid is the unmineralized, organic portion of the bone matrix that forms prior to the maturation of bone tissue. When there is insufficient nutrient, minerals or osteoblast dysfunction, the osteoid does not mineralize properly, and it accumulates. Masson’s trichrome stains osteoid in pink-orange-red.

ROIDefine or transpose the ROI as previously described

Osteoid Thresholding/Editing.- In the “Selected” box of the “Selected List” region, click D13 to make “Osteoid

Volume” the current array.- In the “Large Image Navigator” use the “1:1” ratio button to display a high magnified

view in the “Image Window”.- Place the “Pan Box” in the top left corner in order to scan systematically your section

for osteoid.

Automatic thresholdingo To be able to see the range of colors taken into account by your threshold

the button “Show” has to be active. o Click the “Select” button in the “Threshold” region.o In the “Image Window”, click on mineralized bone. You can use the “Z” key

to zoom in. Continue to click on other regions of mineralized bone. Each click will add the selected color to the range that identifies mineralized bone. It is better to underestimate the thresholding and use the editing tools to fix it.

o To add a large region of mineralized bone, click and drag the cursor. All the selected colors will be added to the range that identifies mineralized bone.To undo, press the “U” key.

o To exit the Image Window, right click.

Manual threshsoldingAs the osteoid surfaces are usually stained in pink-orange-red shades that could also be aspecifically represented in the tissue, manual thresholding can be helpful.

o In the “Threshold” region, click the “R”, “G”, “B” button to turn off the automatic thresholding.

9

o In the “Editing” region, click “Draw Threshold” icon, and in the “Image Window” draw the osteoid surface. Use the zoom (Z key) and resize cursor (scroll) as needed.

o When you are done, right click to exit the “Image Window”.o In the “Editing” region, click the “Erase” icon and clean the hedges of the

osteoid surface. Use the zoom (Z key) and resize cursor (scroll) as needed.o When you are done, right click to exit the “Image Window”.

Osteoid Measurement.- In the “Measurement” region make sure that:

o “Type” is set to “Osteoid”o “Width” is assigned to I1 arrayo “Surface” is assigned to L1 arrayo “Volume” is assigned to D13 array

- In the “Measurement” region, click “Preview” button.- In the “Image Window”, click on the osteoid area. A yellow preview tracing appears

around the osteoid surface.- In the “Measurement” region, click “Set Ends” button.- In the “Image Window”, click on the osteoid area, where the osteoid seam begins

and ends. Yellow X’s appear and green lines show up illustrating the thickness of the osteoid. Red X’s indicate the osteoblast side of the osteoid surface. If they are not on the correct side, click the “Reverse” button in the “Measurement” region.

- In the “Measurement” region, click “Measure” button; “Calculations” and “Overview” update.

- In the “Calculations” window, the 5 parameters will be measured or calculated are displayed on the “A” channel.

- If you have made a mistake, go to the “Edit” menu and select “undo auto object measurement”.

- Once the scan of the ROI is completed, Go to “File” menu and select “Save data”.

10

OSTEOBLAST MEASUREMENTS: N.Ob, N.Ob/BS

ROIDefine or transpose the ROI as previously described

Osteoblast Measurements.- In the “Selected” box of the “Selected List” region, click D15 to make “Osteoblast

Number” the current array. In this array no color thresholding is required.- In the “Editing” region, click “Draw Threshold” icon. The cursor enters the “Image

Window”.- In the “Image Window resize the cursor (scroll) to about half the size of an

osteoblast. Click once on each osteoblast along the osteoid. Use the zoom (Z key) and resize cursor (scroll) as needed. Right click to exit the “Image Window” once you are done.

- In the “Measurement” region make sure that:o “Type” is set to “General”

- The dots you put on the osteoblasts have yellow preview outlines.- At his point, if you want to start over, on the Threshold region, click Refresh to erase

all thresholds.- In the “Measurement” region, click “Measure” button.- “Calculations” and “Overview” update. If you have made a mistake, go to the “Edit”

menu and select “undo auto object measurement”.- In the “Calculations” window, the 2 parameters will be measured or calculated are

displayed on the “A” channel.- Systematically move the “Pan Box” in the “Large Image Navigator” in order to scan

the entire surface of your section and repeat the above steps from “Osteoid Thresholding/Editing”.

Comment: If you plot anything outside of the ROI, it won’t be recorded.

Once the scan of the ROI is completed, Go to “File” menu and select “Save data”.

11

OSTEOCLAST RELATED MEASUREMENTS: Oc.S, Oc.S/BS, N.Oc, N.Oc/BS

Tartrate-resistant acid phosphatase (TRAP) staining is classically used as marker for osteoclasts

ROIDefine or transpose the ROI as previously described

Threshold for TRAP staining (osteoclasts)- Activate L2 array in the “selected” menu within the “Selected List” region.- To be able to see the range of colors taken into account by your threshold the

button “Show” has to be active. - Click the “Select” button in the “Threshold” region.- In the “Image Window”, click on TRAP stained region. You can use the “Z” key to

zoom in. Continue to click on other regions of TRAP positive areas. Each click will add the selected color to the range that identifies positive TRAP staining. It is better to underestimate the thresholding and use the editing tools to fix it.

- To add a large region of TRAP staining, click and drag the cursor. All the selected colors will be added to the range that identifies osteoclast.

- To undo, press the “U” key.- To exit the Image Window, right click.- If you want to start over, press the “Select” button in the “Threshold” region and

repeat.- In the Threshold region, click the Assign button to assign the threshold to the L2

array.

Refining- In the “Measurement” region, ensure that “Type” is on “Auto Surface”.- Click “Preview” button. TRAP positive cells threshold and the preview tracings of the

osteoclast surfaces appear in yellow lines.- In the “Measurement” region, adjust “Minimum Size”, if necessary. The default

minimum surface size is 15. Adjust this value based on the visual feedback of the preview outlines. If this value is changed, you have to be consistent across the different specimen for the all study.

- Preview again to update.- Then, in the “Measurement” region click “Assign” to assign this value to the L2

Osteoclast Surface array.

12

Editing the threshold- In the “Selected List” region, in the “Selected” list, make sure that L2 “Osteoclast

Surface” is the active array.- In the “Large Image Navigator” click 1:1 button so that the highest magnification is

displayed within the “Image Window”.- Starting from the upper left corner of the Tissue Volume ROI, scan systematically

looking for Osteoclasts.- In the “Editing” region, click “Draw Threshold” icon, and in the “Image Window” fill

any TRAP staining that should be thresholded but is not. Use the zoom (Z key) and resize cursor (scroll) as needed. When you are done, right click to exit the “Image Window”.

- In the “Editing” region, click the “Erase” icon and use it to clean the erroneous thresholded areas or separate touching osteoclasts. Use the zoom (Z key) and resize cursor (scroll) as needed. When you are done, right click to exit the “Image Window”.

Osteoclast Measurement- In the “Measurement” region click the “Measure” button.- “Calculations” and “Overview” update and yellow temporary lines will turn

permanent green.- In the “Calculations” window, the 4 parameters will be measured or calculated are

displayed on the “B” channel.- Continue to navigate through the entire ROI and repeat from the editing step.- Once you have analyzed the all ROI, from the “File” menu, choose “Save Data”.

13

FLUORESCENT LABELING MEASUREMENT: sLS, dLS, MS, MS/BS, Lr.L.Wi, MAR, BFR/BS, BFR/BV, BFR/TV, Omt.

Mice are given 2 injections of calcein, normally 7 days appart, although this interbval may be shorter in young animals. The goal is to measure the percentage of the bone surface that is mineralizing and the distance between the labels created by the two injections.

ROIDefine or transpose the ROI as previously described

Tissue volume measurement- In the “Selected List” region, in the “Selected” list, activate the L4 array. - In the “Measurement” region, ensure that “Type” is on “General”.- Click “Preview” button. The entire Tissue Volume preview outlines turns from light

green to yellow - Then click “Measure”. The ROI turns from pink to light green and the preview outline

changes appears in dark green in the “Overview Window” (indicating the effective measurement).

- If you make a mistake, from the Edit menu, immediately choose “Undo Auto Object Measurement” to revert the system to its state before the Measure button was clicked.

Threshold for sLS- In the “Large Image Navigator” click 1:1 button so that the highest magnification is

displayed within the “Image Window”.- Starting from the upper left corner of the Tissue Volume ROI, scan systematically

looking for sLS and.- In the “Measurement” region, ensure that “Type” is on “Auto Surface”.- Click “Preview” button. Fluorescent positive surfaces tracings are generated (yellow

lines)

Editing the threshold for sLS- In the “Editing” region, ensure that the “Type” is set to “Threshold”, click “Erase

Threshold” icon, and in the “Image Window” erase any thresholded object that is NOT single label including double labeled surface. Use the zoom (Z key) and resize cursor (scroll) as needed. When you are done, right click to exit the “Image Window”.

14

- To add threshold, in the “Editing” region, click “Draw Threshold” icon, and in the “Image Window” draw the object you want to include in the threshold.

sLS Measurement- In the “Measurement” region click the “Measure” button.- “Calculations” and “Overview” update.- Parameters that will be measured or calculated will be displayed on the “C” chanel

of the calculations window.- If there is no double labeled surface in this field of view, continue to scan next fields

for labeled surfaces.

dLS Measurement- In the “Selected List” region, choose L5 to make “Double Labeled Surface” the

current array. Selecting L5 also turns off the thresholding. - In the “Measurement” region, “Type” automatically changes to “dLS”. Also ensure

that:o Width array is set on I2o Surface array is set on L5o Spacebar box is checked

- In the “Measurement” region, click the “Draw” button, the cursor enter the “Image Window”.

- Click along the outer label in the double labeled surface. Stop when there is no inner

label opposite to the surface. Tap the spacebar to end the outer label tracing.- Then, click along the inner label opposite the outer labeled surface. Tap the

spacebar to end the inner label tracing and right click to exit the Image window. The outer and inner labels turn pink and yellow respectively. Light green lines appear going from one label to another.

- In the “Measurement” region click the “Measure” button.- “Calculations” and “Overview” update.- Parameters that will be measured or calculated will be displayed on the “C” chanel

of the calculations window.- Repeat the dLS procedure for all double labeled surfaces in this field of view.- Continue to navigate through the entire ROI searching for labeled surface.- Once you have analyzed the all ROI, from the “File” menu, choose “Save Data”.

15

Data Exportation- From the File menu, choose Data Manager. The Data Manager opens.

- In Data Manager, use the left panel to navigate to the folder where your project is stored. Click to highlight the folder. Your data is stored in C:\BQDATA\Your Name.

- In Data Manager, click the Show Volumes button. The middle panel fills with Data Volumes.

- In the middle panel, click to highlight the Data Volume for the Project.

- In Data Manager, on the toolbar, choose the Export Data Table button.

- On Export Data Table click the Add button to add the highlighted Project Data Volume’s data sets to the Data Sets in Table box.

- Click the To Excel button.

- On the Save Data Table to Excel box, navigate to where you would like the Excel file saved. Then type a File name and click the Save button.

16