Workflow

CellX is an open-source software package of workflow template for cell segmentation, intensity quantification, and cell tracking on a variety of microscopy images with distinguishable cell boundary.

Installation and step-by-step usage details are described in Mayer et al (2013). 

After users provide a few annotations of cell sizes and cell boundary profiles, it tries to match boundary profile pattern on cells thus provide segmentation and further tracking. It works the best on cells without extreme shapes and with a rather homogeneous boundary pattern. It may not work well on images with cells of sizes only a few pixels. Its output comprises control images for visual validation, text files for post-processing statistics, and MATLAB objects for advanced subsequent analysis.

The Fiji distribution of ImageJ comes with several manual tracking tools, two of which are particularly useful:

* _Plugins->Tracking->Manual Tracking_

* _Plugins->Tracking->Manual tracking with TrackMate_ (TrackMate is an advanced automatic tracking tool, with the option for manual editing of tracks)

The _Manual Tracking_ plugin is quick to use, intuitive and produces easy-to-understand output. TrackMate has the advantage that automatic detection and linkage can be combined with manual input.

Update sites

MtrackJ (see the component page here) can be installed via Fiji update sites. It has many shortcut keys enabled so for manually tracking many data, it will become quite efficient as you get used to the short-cut key operation.

Pre-processing

Pre-processing steps before manual tracking might include:

* denoising and/or deconvolution

* flicker and photobleaching correction, e.g. using Fiji's _Image->Adjust->Bleach Correction_

* flat-field correction, and/or bandpass (ImageJ's _Process->FFT->Bandpass filter_) according to the size of the features of interest

The QuimP software from Bretschneider group is deployed as ImageJ plugin and includes model-based cell segmentation, cell outline tracking and quantification of the spatially resolved speed of protrusions and retractions. The algorithm to calculate morphological dynamics is faster compared to other approaches (e.g. Machacek and Danuser, 2006). The reference paper describes the workflow for these analyses.