ROIs can be created and edited using commands gathered in the ROI menu. ROIs can be also created from binary objects, graticules (for equally spaced shapes) and from binary layers created by tracking.

Figure 365. Available ROI tools

Individual ROIs (ROI objects) are 2D objects. They can have different shape: rectangular, elliptical polygonal or Bezier. One shape can be created with one or more tools. For instance polygon ROI can be defined using both “Draw Polygonal ROI” and “Autodetect ROI”.

In case of an ND file which has time axis (e.g. time-lapse) individual ROI objects can be defined as “Global ” or as “Changing over time”. Global objects do not change any of their characteristics over time. Objects that are defined as changing over time can change their position and shape in time (however objects cannot change their shape - e.g. from ellipse to polygon). This feature is useful for track moving or warping objects.

To track moving objects use the “Edit ROIs in Time” editor or just change any global ROI into changing over time from the objects' context menu and move it or change its' shape (see interactive edit) on two or more frames. Such frames are called “key-frames” (2 shows trajectories with little boxes representing the key-frames). All frames in between key-frames are interpolated from these two key-frames. Frames before and after first and last frame respectively are taken from that first or last frame. Therefore it is not necessary to reposition or redraw the ROI on every frame.

In case of Multi Point ND2 file there is one more option: ROI can be defined per Multi Point (which is the default). It means that ROIs from one point are completely independent of ROIs defined on the other point (even if they have the same number). This applies also to ROIs defined as “Changing over Time” (in case the image has time axis too). On the other hand if the ROI is changed to a “Global” it will be shared among all points of the Multi Point set.

Depending on the application purposes ROI objects can have different usage. NIS-Elements can handle “Background ROIs”, “Reference ROIs”, “Stimulation ROIs” and “Standard ROIs”.

ROIs can be manipulated anytime directly in the image window. Many of the following techniques work in ROI editors too. Objects cannot be moved or resized when they are Locked (some analysis tools or users may lock them to prevent accidental modification). They must be unlocked (from the ROIs' context menu) before any manipulation. Note that the mouse cursor changes over the ROI to indicate which action will be started after mouse button is pressed (it changes when modification keys like Alt and Ctrl are pressed).

Exact values can be entered in “ROI Properties” dialog which is accessible from the ROI context menu.

Figure 366. 

When creating more than one ROI, the Simple ROI editor is the tool of choice. With this tool it is possible to make all possible shapes with different methods. All interaction techniques work here as well.

Figure 367. Simple ROI Editor toolbar

Note that many actions have a hot-key (P for Pointing tool, R for Rectangle, E for ellipse, L for Polygon, B for Bezier, A for Auto detection, H for drawing holes and others) to speed up the process of interaction.

Auto-detect tool is available from ROI menu, Simple ROI editor, Edit ROIs in time editor and some other places. It produces a Polygon ROI. Using the tool is two step process: The first click on an image structure results in preliminary object contour, that is drawn on the screen. After that, user can further tweak the object contour and then finish whole auto-detect by confirming with right mouse button. User can always cancel auto-detect by pressing the Esc key.

The very first click is important, because these pixels under the cursor determine intensities from which the algorithm guesses the whole object. As a general rule it is better to choose brightest pixels in the bright objects (fluorescence) and dimmest in the dark object. It is also good to click near the center of the object.

ROIs are used mainly in the following procedures:

In all these cases ROI selection is taken into account as well as intersection options in Measurement Options.

Additionally the Time measurement interprets the ROI usage type. Background ROIs are used for offset subtraction and Reference ROIs can be used for bleaching correction.

The Edit ROIs in time editor simplifies editing ROIs that are changing over time. Run ROI > Edit ROIs in Time to display it. ROIs created and edited with this tool are converted into “changing over time”. All operations can be rolled back by Undo/Redo.

Figure 368. Edit ROIs in Time toolbar

The editor has two major tools: Draw and Detect. Both produce polygon shapes but in the first case it is drawn manually around a structure on the image whereas in the latter it is automatically detected after one click. When Draw or Detect is selected, it is possible to start drawing or detecting immediately as indicated by cursor symbol. In case of editing an existing object, it will be hidden after this first click is made, so the new one can be drawn unobstructed. Esc key is always available to cancel and revert to the previous state. Other tools are for Picking, Moving and Deleting objects.

In this editor, there is always exactly one object selected. Its name and color is reported in the first combo box. In the image here reported, there is object 1 (red) currently selected. To toggle between creation of new objects and editing of existing ones there is the New button.