Track and Lineage Construction – Algorithms

The ImarisLineage module includes the full set of algorithms provided with ImarisTrack integrated into the object “Creation Wizard” of Imaris for “Spots Objects”, “Surface Objects” and “Cell Objects”. All of the tracking algorithmscan be applied to 2D time series or 3D time series (4D) images. Additionally, ImarisLineage includes the ability to automatically detect and track cell divisions. Cell fusions are also automatically detected and tracked. The Lineage algorithm is based on the AutoRegressive Motion algorithm which automatically adjusts to changes from the initial distance constraint (acceleration / deceleration).

Visualization

The tracking results in ImarisLineage are presented in two windows:

• Track editor - as a 2D lineage tree displaying all objects connected with tracks for all time points
• Surpass 3D view - as an exact path of the object in 3D including cell division event

The two views remain interactive as selecting objects, tracks, lineages and branches in the track editor selects the same group of objects in the Surpass view. Also editing one view changes the other.

Track editor in ImarisLineage includes several new visualization and presentation tools such as:

• Spatial organization - the lineage tree can be presented vertically or horizontally and the branches can be flippedas needed. Tracks can be moved left or right to ensure optimal presentation of the lineage tree.
• Labelling - groups of objects in the lineage tree (as segments, branches, tracks) can be selected and labelled with colors according to the user’s need (i.e. progenitors coming from the same mother cell) and the same color visualization appears in the Surpass view.
• Color coding - within the track editor both tracks and points can be color coded according to any chosen statistical value (just like in the Surpass view), i.e. fluorescence intensity, volume, generation.

Surpass view in ImarisLineage includes all display and visualization modes known from ImarisTrack and as well as additional visualization tools specific for dividing objects, among them:

• Track Display – Directly visualize the track as an exact path of the object ( tracks split after a cell division), using a line for high contrast, or a shaded cylinder for enhancing relative depth perception. On the tracks you can visualize objects, the center point of the object, or just the track alone when the objects might obscure the view.
• Color Coded Tracks and Objects – Tracks and objects can be color coded according to any specific statistical values (in combination with Imaris MeasurementPro) or may be color-coded based on time. ImarisLineage adds color coding based on Generation, Cell Cycle Duration, Track number of generations, Track number of branches or Track position start
• Labelling Objects - Objects such as Spots, Surfaces and Cells can be displayed in different colors based on Labels which are assigned to groups of objects by the user (e.g. branch of a lineage tree). Users can also visualize one or more labelled groups of objects at a time (e.g. to better inspect the spatial distribution of cells coming from the same mother cell).
• “Dragon Tail” of Tracks – Customize the length of the track path for visual clarity by displaying the path as a dragon-tail which only shows the selected number of time points.

Measurements

The goal of tracking objects is to either measure how they change over time, their motility behaviors, or their cell cycle characteristics. ImarisLineage, in combination with Imaris MeasurementPro, provides a variety of measurements to facilitate analysis.

• Cell Cycle Specific Statistical Values - Generation, Cell Cycle Duration, Time since first division, Time since previous division, Normalized Time since previous division, Track number of generations, Track number of branches
• Track Specific Statistical Values – Duration, length, straightness, displacement X, Y, Z and total, squared displacement, velocity X, Y, and Z, average speed, instantaneous speed, speed variability, quality of fit to autoregressive model (short-term predictability) - per-axis or mean of all axes.
• Object Time Dependant Statistic Values – Changes in size, shape and intensity over time. In addition to being displayed in a table, these statistics are shown as a graph. You can select a time point of interest in the graph and the visual view in surpass changes accordingly.
• Filtering, Sorting, Classifying and Grouping – Using the interfaces described for Imaris MeasurementPro, tracks may be filtered sorted, classified and grouped based on the track specific parameters.

Track statistics can be exported as .csv, Excel .xml or native Excel .xls files. The statistics can be exported for one track, a group of tracks, or all tracks. Additionally, statistics can be exported for one, a selection or all measured parameters. Finally, it is possible to specifically export the data needed to replicate any of the interactive plots Imaris creates/shows. The statistics of all segmented objects associated with a track can also be exported.

Editing

ImarisLineage offers a set of tools to modify tracks once they have been created. This feature allows manual creation of tracks and lineage trees, editing of the automatically generated lineages, or for the correction of drift in parts of the image that are moving that should not be.

• Translational and Rotational Drift Correction – ImarisLineage can track all objects in an image, even objects that should be stationary. Once tracked, an object or series of objects that should not be moving can be selected. Then positions of all other objects within the image are corrected based on this anchored object or average of the selected objects. This positional shift results in drift correction in the image and the anchor object(s) expected to be stationary will be stationary in the resulting image. The drift correction allows the detection of the true movement of the other objects of interest. In addition to translational drift correction, rotational drift correction can be utilized if at least three objects in various parts of the 3D image that should not be moving are selected as thebasis for drift correction.
• Track Editing – Sometimes automatic tracking is not perfect and researchers need a system to correct for errors. ImarisLineage makes track editing quick and easy. Within the Surpass view or Track editor you can select two objects in subsequent time points and connect or disconnect them to modify the track. You can also connect daughter cells to the mother cell or connect two or more objects after fusion. For more streamlined workflow in the track editing, moving between frames can be done using ←→ arrows.