1. Calibration & Measurements

2. Calibrating the System Before we can make any measurements we need to calibrate our imaging system to create real world numbers. With a Microscope we can calibrate for each objective using a stage micrometer. With any other system we can use any object of fixed length.

3. Calibration First Snap an image of a graticule or and object of known distance. This function is held under the drop down menu Measure/Calibration/Spatial. This will bring up the spatial calibration dialog box.

4. Calibration Procedure Procedure Name your calibration file Select the units you wish to use. Select the Image button- This will place a line onto our image Stretch the line over your known distance. Enter the known distance When happy press OK

5. Selecting an existing calibration Our new calibration can now be seen on the bottom right of the Workspace preview. It will also be save with the image. Once we have made your calibration we can reload it or select another calibration file.

6. Using a Marker If we wish to show our calibration we can by selecting the Marker button. This will bring up the below dialog box.

7. Types of Measurements Manual Tagging - Tagging points of interest for counting Manual Measurements - Lines, circles, widths, angles etc Threshold Based Measurements - Object area, density, % area etc Caliper Tool - Line profile based measurements NOTE : All measurement data can be exported to Excel or ASCII for statistical analysis.

8. Manual Tagging Manual Tagging is used for - Quick object counting of one or more class - Recording XY positions of objects (can be used for tracking objects) - Recording density information.

9. Manual Tagging

10. Manual Measurements These measurements can be located in the Measurement File Menu These measurements are made by user interaction with the cursor on the image to create lines, circles, width etc. These are very easy to use and very quick to get information.

11. The Measurements Dialog Box

12. Dynamic Data Exchange In Image-pro wherever you can generate numbers, there is also an option to export the numbers to Excel. You will find an option for DDE in different places depending on the dialog box the numbers are held in. They reside either under a File drop down menu or on an Input/Output tab. The setup options for DDE are shown opposite.

13. Thresholding As we saw in the first section, objects with differing intensity can be picked out by using the bit maps, line profiles and surface plots. As objects we are looking to select have the same range of gray scale values we can use the information in the Histogram to select objects from the image.

14. The Count/Size Dialog Box A large amount of information can be obtained from this one dialog box. The general count procedure is as follows: Set up options Select Ranges Select Measurements Select to veiw measurements

15. Setting up our options Here we can select the outline and label style. My personal settings are shown but you may choose yours differently based on your application and personal preferences. Once we have entered our own settings we can click OK and return to the Count/Size Dialog box. When happy Click OK

16. Selecting our ranges Next we will need to select our threshold. In rare situations where we have high contrast we can select to automatically pick bright or dark objects. Usually we have to select manual and then Click on Select Ranges. Here we can use the slider bar to threshold in the regions we require as shown on the below image. When happy with threshold click OK.

17. Thresholding As we adjust the threshold parameters on the slider bar we can see objects selected by colour. When all is done and you have selected the threshold we will return to the Main Count/Size dialog box and Select Count.

18. Selecting Measurements Now we may wish to select the measurements we want to make. To do this go to Measure/ Select measurements – this opens the below window. On the left are all the measurements we can make and by highlighting them we find the measurement move into the filter range box in the middle. On the right hand side we find a brief description of what we are measuring. Click OK

19. Viewing Measurements To view all measurements we must select the View File menu from the Count/Size dialog box. Select the option Measurement Data. This will open the Measurement Data Dialog box.

20. Thresholding Colour Images One problem we often find is that colour images are difficult to threshold using the slider bar as we have overlapping colour information, as shown opposite A different approach to selecting a region from the Histogram is to use a Colour Cube based approach.

21. The Colour Cubed Approach Using the eyedropper tool we use the cursor to click on the colour we wish to threshold. When you are happy with the threshold click close. This usually yields better results than thresholding using the slider bar. The measurements are obtained in the same way as for monochrome images.

22. Multiphase Thresholding We can select to threshold more than one phase. For example in the images opposite there are 3 distinct phases and you may want to gather information about each phase. This is mainly done to give % phase informaion.

23. Thresholding more phases To enter a second class click on the New button after you are happy with your first threshold. Ensure in Options that Filled Objects is unchecked. Range statistics can be found under View File Menu of the Count/Size dialog box.

24. Caliper Tool The Caliper Tool adapts the line profile to allow for multiple lines and multiple measurements. As we can see opposite we generated a line across an object and we have rising and falling points of inflection.

25. Setting Detection Once we have drawn our line or lines we select New. This allows us to select to detect Peaks Valleys Rising points of inflection Falling points of inflection

26. Caliper As you can see on the image opposite, I have detected the rising and falling edges so as to measure the length of the object which falls along my line.

27. Caliper Measurements To make measurements move to the Measurements Tab. Select Measurements This allows us to measure between detected feature of one or more type.