The overlays module allows you to visualise the output of tracking and post-processing in the context of the raw images that form your dataset. This allows you to visually verify the accuracy of your data processing, and can also provide novel insights into spatial and temporal structure in your dataset.
Upon clicking the Overlays button on the home panel, you will be presented with the following GUI:
The viewport lets you see how the current overlay settings look when applied to a single frame. Clicking Run 'em all! will then apply these settings to all the frames in your dataset, saving them in a new directory from which they can be loaded and turned into a movie.
The main overlay settings are contained in five drop-down menus:
In addition to these menus, two checkboxes are available at the bottom of the settings panel: Show ID? and Mark events?. With Show ID selected, a small label will appear next to each object containing its track ID. Mark events? allows you to display externally defined events. For further details on how to assign events, please see the advanced usage page.
Once you are happy with the setup of the overlay, click Run 'em all! to apply these settings to all the frames in the dataset. When this has finished, you will be able to access the set of generated frames in the new 'Overlays' folder in the root directory. You can now generate movies from these frames. There are many ways to do this, but two example methods are described below.
FIJI provides a number of utilities for producing movies. A simple method for generating .AVIs is described below.
Further details of the image sequence import method can be found in this Youtube tutorial.
Once you have generated a basic .AVI, tools such as FFmpeg can be used to compress the movie or convert it to a different format.
Movies can also be exported using Quicktime Pro, allowing the user to take advantage of Quicktime's built-in export system to natively generate many different movie formats.
The above steps can be combined to give a wide range of different outputs. Below are some examples of the types of movie that can be generated using the overlay system.
In this movie, the tracks of cells within a high-density 2D system are reconstructed and overlayed on top of the original phase-contrast images. Track sections are coloured according to the time between the start of the track and the time the section was measured, varying between 0 (cyan) to 100 seconds (magenta), the total length of the movie:
In this movie, separate lineages assigned using division detection are indicated with different coloured outlines. Track IDs are also shown:
The 'generational age' of each cell in this dataset is also automatically calculated in the division detection module. In this video, generated using the special 'Lineage trees' option, cell ages are indicated by the colour of the overlying track, starting from generation 1 (dark blue) and finishing at generation 6 (dark red).