BIOQUANT PROTOCOLS
INTRODUCTION 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
Editing the threshold for sLS 13
sLS Measurement 14
dLS Measurement 14
Data Exportation 15
INTRODUCTION
Bioquant interface description
The 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 Guide
o Image
o Selected List
o Parameters
o ROI Tools
o Threshold
o Editing
o 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.
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”.
- 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.
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 ROI
The 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).
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.
ROI
Define 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.
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”.
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.
ROI
Define 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 thresholding
o 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 threshsolding
As 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.
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 array
o “Surface” is assigned to L1 array
o “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”.
OSTEOBLAST MEASUREMENTS: N.Ob, N.Ob/BS
ROI
Define 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.