# Measurements & regions of interest

Chapter outline
• Regions of Interest (ROIs) can be manually drawn and measured

• The accuracy of measurements will depend upon the Image Properties being correct

• Multiple ROIs can be added to the ROI Manager or an overlay

• ROIs can be inverted or combined to give more complex shapes

## Measuring images

The main command for measuring in ImageJ is found under  ▸ Measure (or just press M), where  ▸ Set Measurements…​ determines what measurements are actually made. Possibilities include areas, perimeters, lengths, and minimum, maximum and mean pixel intensities (here referred to as 'gray values'), as well as further measurements of shapes or intensities (Figure 1B). By default, measurements are made over the entire area of the currently-selected 2D image slice, and added to a results table (Figure 1C).

Important!

Size measurements are automatically scaled according to the pixel sizes in  ▸ Image ▸ Properties…​, so these must be correct!

 A: Image B: Set Measurements dialog C: Results table

Figure 1: Measurements made on an image are added to a results table. The choice of measurements to make can be changed using the  ▸ Set Measurements…​ command.

Results tables & their titles

A small idiosyncrasy to be aware of is that, as far as ImageJ is concerned, there is only ever one 'official' results table – the one with the title Results. Different, similar-looking tables can be produced (perhaps by duplicating the official table, or internally by some other command), but any new measurements you make with the Measure command will only be added to the official table. The official table also has an extra Results entry in its menu bar.

 New measurements will be added here New measurements will not be added here
Caution with measurements

Set Measurements…​ deserves your close attention! Because all new measurements are added to the same results table, when working with multiple images it can be hard to remember which measurement refers to which image. It is therefore a very good idea to choose Display label under Set Measurements…​, to ensure the image title is included in the table. When working with higher dimensions, choosing Stack Position lets you know which 2D slice of the entire dataset has been measured.

Also, for now, make sure that Redirect to is set to None. This is normally what you want, to avoid merrily measuring the wrong image by accident (see Detection by thresholding).

## Regions Of Interest

Usually we want to measure something within an image and not the whole thing. Regions Of Interest (ROIs) can be used to define specific parts of an image that should be processed independently or measured, and so only pixels within any ROI we draw will be included in the calculations when we run Measure.

 A: Tool bar B: Image + ROI

Figure 2: ROI drawing tools are found on the left side of the ImageJ tool bar (A). The ROI in (B) was created by drawing one rectangular and two circular ROIs, holding down the Shift key between each so that the regions were combined.

ROIs of different types (e.g. rectangles, circles, lines, points, polygons, freehand shapes) can be drawn using the commands in the tool bar (Figure 2), and are invariably 2D. Right-clicking the tools often provides access to related tools, while double-clicking may give additional options. When drawing a ROI, pressing Shift or Control before releasing the mouse button adds the ROI being drawn to any existing ROI already present.

Some extra commands to create or adjust ROIs appear under the  ▸ Selection submenu, which we will make more use of later.

Measurement accuracy

Although ImageJ can measure very exactly whatever regions it is told to measure within an image, keep in mind that in light microscopy images any size measurements will not exactly correspond to sizes of structures in real life. This is especially true at very small scales (hundreds of nanometers or smaller), for blur-related reasons that will be described in Blur & the PSF.

## Working with multiple regions

Normally, only a single ROI can be 'active' (i.e. affecting measurements) at any one time. If you need control over multiple ROIs, there are two places in which you can store them, differing according to purpose:

1. The ROI Manager: for most ROIs that you want to be able to edit and use for measurements

2. The image overlay: for ROIs that you only want to display

### The ROI Manager

The ROI Manager provides a convenient way to store multiple ROIs in a list, allowing you to easily access, edit and measure them. The slow way to open it is to choose  ▸ Tools ▸ ROI Manager…​. The fast way is just to draw a ROI and press T[1]. The additional Measure command within the manager is then like applying  ▸ Measure to each ROI in turn. If you happen to want to show all the ROIs simultaneously, you can select the Show All option[2].

Because ROIs in the ROI Manager are represented independently of the image on which they were defined, you can create a ROI on one image, add it to the ROI manager, select a different image and then click on the ROI in the ROI Manager to place it on the second image. Measurements made from the ROI Manager always use the most recently-selected image, so be careful if you have several images open at the same time.

Transferring ROIs

A faster way to transfer ROIs between images without using the ROI Manager is to click on the second image and press Shift+E (the shortcut for  ▸ Selection ▸ Restore Selection)

Expert ROI manipulation with the ROI Manager

Using the ROI Manager, you can craft your ROIs into more complex shapes, adding or removing other ROIs.

To do this, first add the main ROIs you want to work with to the manager. Then select them, and choose from among the options:

• AND – create a ROI containing only the regions where the selected ROIs overlap

• OR – create a single ROI composed by combining all the selected ROIs

• XOR – create a single ROI containing all the selected ROIs, except the places where they overlap ('eXclusive OR')

### Overlays

Overlays also contain a list of ROIs that are shown simultaneously on the image, but which do not affect the Measure command. They are therefore suitable for storing annotations for visualization purposes. You can think of them as existing on their own separate layer, so that adding and removing the overlay does not mess up the underlying pixel values (Figure 3). The relevant commands are found in the  ▸ Overlay submenu, where you can get started by drawing a ROI and choosing Add Selection (or simply press B[3]). The same submenu also provides commands to transfer ROIs between the overlay and the ROI Manager.

You can 'reactivate' a ROI on an overlay by clicking it with the Alt key pressed (provided a suitable ROI tool is selected).

### Saving ROIs

Individual ROIs can be saved simply by choosing  ▸ Save As ▸ Selection…​ The ROI Manager itself has a Save…​ command (under More), which will save whichever ROIs are currently selected (or, if none are selected, all of them). Overlays are fixed to specific images and do not have their own special save command, but will nonetheless be included if you save the image as a TIFF file (ImageJ’s default format). Any currently-active ROI will also be saved in a TIFF.

This is fine if you work only in ImageJ or Fiji, but unfortunately if you try to view your ROIs in other software it is highly unlikely to work properly, since the format is specific to ImageJ. The way around this is to use the  ▸ Overlay ▸ Flatten command. This creates an RGB copy of the image in which the pixel values have been changed so that any ROIs or overlays will appear whenever you open the image elsewhere. Therefore you may well want to use this command when creating figures or presentations, but you do not want to subsequently apply your analysis to the image you have flattened – always use the original instead.

For more details on the impact of converting an image to RGB, see Channels & colors.

Figure 3: ROIs and overlays are displayed on top of images, and so can be removed easily without having any effect upon the pixel values. Flattened images may appear the same on screen, but are invariably RGB (see Channels & colors) and have had their pixel values permanently changed to show any annotations.

Practical

Open the images Annotated_Cyclists_1.tif and Annotated_Cyclists_2.tif, which depict the 3 main cyclist characteristics I found most disconcerting as a pedestrian in Heidelberg.

The images should initially look the same, but in one the text is an overlay, while in the other the image has been flattened. Which is which? Try to think of several ways to investigate this.

Annotated_Cyclists_1.tif is the one with the overlay.

Five ways to determine whether an annotation is an overlay or not:

1. Zoom in very closely to the region containing the annotation. If it becomes 'blocky', i.e. made up of pixels, it is not an overlay. If it remains smooth, then it is.

2. Move your cursor over the region where the annotation appears, and look at the pixel values. If the values are all the same where the annotation is present, but different elsewhere, then it is unlikely to be an overlay.

3. Using the paintbrush or pencil tool from the toolbar, try putting some other color where the annotation appears. If the annotation remains visible on top of where you drew, it must be on an overlay.

4. Choose  ▸ Overlay ▸ Hide Overlay and see if the annotation disappears.

5. Choose  ▸ Overlay ▸ To ROI Manager and see if anything happens at all.

Practical

Using the cyclist image containing the overlay from the previous practical, rearrange the annotations so that they are each positioned next to different cyclists. You could do this by deleting the overlay and starting again, but there are other, faster possibilities (using a technique mentioned before, or the  ▸ Overlay ▸ To ROI Manager command).

Solution since ImageJ v1.46m: Click the annotation while holding down the Alt key, to bring it to life so it can be moved around again. This only works if certain tools are selected, e.g. Rectangle or Text, because some other tools have more overriding functions, such as zooming in or scrolling.
2. If you have a stack, you also may need to explore More >> Options…​ to define whether all ROIs are shown on all slices, or only on the slices on which they were first created.