Open the software by executing ExTrack_GUI.exe in the folder ExTrack_GUI.

Data loading

The first window displayed by ExTrack_GUI is the following: In this window, the user must inform the localition of the data to analyse. In the path box, either indicate the name of the csv file to analyse or the name of a folder that contains csv files. If a folder is indicated, ExTrack will analyse simultanously all the .csv files in the folder. This option is useful when a replicate is composed of several movies that each generated a csv file. NB: The browser can only select files. Therefor, copy/pasting is the most straitforward way to provide a folder path.

Data format

ExTrack requires tracking data as inputs. ExTrack_GUI more specifically uses comma separated values (.csv) format so the first step is to convert your data into this format if that is not already done. In this format, the first line must specify the names of the columns and each of the following lines represent informations on one peak detection. A minimum of 4 lines are required: the x position of the peak, the y position of the peak, the frame of the peak and the track ID (an integer number that is specific to the track that the peak belongs to).

Track length

The user must inform the minimum and maximum lengths of tracks to consider. Tracks with less peaks than the minimum length will not be selected and tracks with more peaks than the maximum length will be cut to their first positions (maximum length). The computation time of the fitting analysis is proportional to the number of track lengths so limiting the track length to a fixed number can considerably speed up the fitting (especially on windows where parallelization is not possible). However, to avoid the defocalization bias, it is best to select the widest range of track lengths as possible (example tracks of length 3 to 50 if the average track length is 10).

Column header names

The default names for the 4 required columns is POSITION_X, POSITION_Y, FRAME and TRACK_ID but these names can be changed by modifying them in their respective boxes in the line headers.

Maximum distance

This parameter allows the user to get ride of tracks with unexpectedly large displacements (step-wise displacements larger than the maximum distance). Set it to a large number if you do not not want to filter out any tracks.

Remove no displacements

If set to yes, this will remove the tracks that possess no displacements. In TrackMate, the peaks at the edges of the image are often detected with fixed positions that are not representative of the true positions so this option can be used to filter out such tracks.

Analysis types

Extrack enables the user to perform 4 types of data analysis:

• Model Fitting: Fit a model to the dataset to estimate the parameters that best represent the data.
• State labeling: For a given model and set of parameters, provides the probabilities of each peaks (each time point of each track) to be in the different states.
• State lifetime histograms: Provides the lifetime distributions for each state.
• Position refinement: Provide better estimate of the positions (and refined errors) of tracks at time steps where the particle is immobile or slowly diffusive without affecting the estimate of fast diffusive particles to optain the best tade-off between spatial and temporal resolutions.

Type of localization error

ExTrack offers 3 options to deal with localization error:

• Fitted parameter: The localization error is considered like a parameter to fit. It is assumed to be the same for x and y and independent of the state. This assumption is good when the movies show moderate variations of peak intensities.
• Imputing the localization error: This option requires to provide an estimate of the localization error for each peak based on the images. The name of the column that provides the localization error must be informed on the right box.
• Imputing a quality metric: This option requires to provide a quality metric for each peak based on the image. That metric must be proportional to the intensity of the peak (and around 0 for background). The name of the column that provides the quality metric must be informed on the right box.

Additional metrics

The state labeling and the position refinement tools save their results in a csv file. If you want to pass additional columns from the original csv file to the resulting csv file, add the names of the columns to keep. If you want to save more than one column, add the different column names separated by commas without spaces (example: MEAN_INTENSITY_CH1,MEDIAN_INTENSITY_CH1).

Next

When all the data information and analysis parameters are entered, click on the Next button.

loading time

If the csv files contain many tracks, the loading process may take some time (1 min).