Using ImagePro Plus

a short walk through Image-Pro Plus 6.0

Image-Pro Plus 6.0 is a powerful scientific image analysis software package. While it can easily be used with existing images it is most often used to capture images directly from a video camera or a digital camera such as the Spot RT which can be mounted on any of our instruments, including our Zeiss microscopes and the Wild M420 macroscope. The basic steps to be followed when using ImagePro Plus are:

1. acquire (or open) the image,
2. enhance the image as needed for measurement,
3. process the image as needed with, for example, edge dection filters,
4. calibrate the software for the scale or magnification of the image,
4. measure or count the structures of interest to you,
5. export the measurements to your spreadsheets, graphing or stats software.

Pressing the Acquire button or selecting the Acquire/Video digital menu choice will bring up the dialogue seen below for the Matrox Meteor II frame grabber, which is used to bring a video signal from an NTSC video camera into the computer or you can choose the SPOT RT-KE camera. The settings on either dialogue tab should not be changed. If you are acquiring images from the microscope press the Start Preview button. Assuming that you have focused the specimen and adjusted the optics of the microscope for Köhler illumination and that you have light going to the camera you should see a live image on the screen. Pressing Snap should then freeze a copy of the frame, leaving it ready for measurement and data extraction.

If you are working with already existing images, File/Open the files that you want to work with. In either case the Image Pro workspace will look something like the screen below after an image has been opened or acquired, with the image on the left and the image capture dialogue on the right.

ImagePro workspace

In addition to drop down menus, there is a tool bar across the top of the workspace which gives direct access to some of Image-Pro's most used functions. Some of these tools are identified in the figure to the left. The Image Database is a utility for cataloging, organizing and finding existing images. Video Capture launches the video capture dialogue seen above. Annotate allows manual labeling of the image.

At the very least, some Contrast Enhancement of images is usually required. The dialogue for this is shown below, in its expanded form. Adjustments can be made either by using the three sliders in the left of the box (brightness, contrast and gamma, from left to right) or, for more exacting control, by shaping the curve in the middle, which adjusts the Look up Table (LUT) that controls the relationship between input greyscale values and output or display greyscale values. Best Fit Equalization spreads the greyscale information across the available range from black to white, merging the bottom 3% of values to black and the top 3% to white. Reset Contrast returns the image to is original contrast.

In addition to contrast adjustment, a wide variety of specialized Filters and Operators can provide further image enhancement. These include a wide variety of edge and detail enhancement strategies, ways of separating structures through Erosion and Segmentation so that they can be counted and methods for subtracting uneven backgrounds from images. Fast Fourier Transformations (FFT) can remove repetitive patterns that obscure image information. Colorization allows manipulation of the LUT to convert the different shades of grey of a black & white image to false colours with, for example, dark grey becoming blue and light grey becoming red.

The Calibrate button allows the correct calibration to be selected for the image's scale or magnification. The calibration is set via the dialogue at Measure/Calibrate/Spatial. Count and Measure allows automated gathering of data with specified parameters and Manual Measurement allows individual manual measurements to be made.

Contrast dialogue

The measurement tool box, as seen below, has tools for selection, deletion, deletion of all, point, line, circle, rectangle, polygon, trace, best fit line, best fit circle, best fit arc, distance between, angle, angle between features, horizontal thickness, vertical thickness and curve thickness. The use of each will allow capture of a variety of data which can later be edited to that which is relevant. When a tool is selected you will be prompted as to how to click in the image to make each measurement. As each measurement is made it will be labeled within the image and the result will appear on the Measurement tab. When finished, these can then be exported to a spreadsheet or stats program via the Input/Output tab. If you need a copy of the picture with its overlay (the circles, lines and lables) go to menu File/Screen Capture and set the parameters in the dialogue box as needed. Then press F7 to capture and save the active image window.

Measure dialogue

The fluorescence image below has a region of interest selected (outlined in blue). In this example, the cells exhibiting orange fluorescence are to be counted.

Fluorescence image

The count/size dialogue has been selected and set to manual. Pressing the Select Colours button launched the segmentation dialogue, as seen below. The eyedropper on the Colour Cube Based tab was used to select the colour range of the cells to be counted. Some images suit a brightness criteria instead of a colour criteria, in which case selection is made via the Histogram Based tab. The dialogue was then closed and pressing the count button produced a count of 261 cells.

count dialogue Segment dialogue

The Image menu on the Count/Size dialogue has an extraction tool under Make Sorted Objects Image that will extract the counted structures and sort them by size, area, length or other factor, resulting, for example, in an image similar to the following.

extracted image

Another useful function in Image Pro is the Process/Extended Depth of Field. It allows the combining of the sharp portions of a number of same sized images. Figures 1-7 below are a series of "optical sections" from our confocal microscope and the final figure is the "combined" image.

In order to get an overview and a better idea of all the menu options and choices available in Image Pro, please have a look at the Image Pro Menu page. In addition to the software's built in functions, macros can be installed to extend its capabilities. Have a look at those that are available at Media Cybernetics' Solutions Zone. For further reading about using ImagePro Plus see the Start Up Manual and the Reference Manual.