TITLE: Easy tracking of unstained cells in ImageJ
AUTHORS: Isabella Gregorski, Henrike Rebl

[DESCRIPTION]

This protocol shows how stained and especially unstained cells can be tracked in ImageJ without much effort.

The aim was to establish a reproducible setup for electrical stimulation and to perform live tracking of the stimulation with a confocal laser microscope. The study also aimed to develop a methodology for a comprehensive and standardised assessment of all parameters independent of cell staining.
The underlying experiment aimed to develop methods for analysing cellular responses to electrical stimulation and to investigate the effects of electrical stimulation on the adhesion and migration behaviour of L929 and HGF-1 fibroblasts using an electrotaxis chamber. This method can be used for single cells and cell clusters and is not limited to fibroblasts. Scratch assays and other image series from the LSM can also be analysed.

For the experiment, time-lapse series were acquired every 5 minutes over 18 hours using an LSM 780 microscope (Carl Zeiss Microscopy GmbH, Germany; software: ZEN 2.3 SP1 FP3, black, Ver. 14.0.27.201). The images were exported to the ImageJ software (1.54f, Rasband, National Institute, USA) to analyse cell migration, which is presented here. Since a bicarbonate-free medium was used, additional CO2 gasification was not necessary.

Slight deviations from the original setup can lead to different results, and insufficient parameter information makes reproducibility difficult. It is, therefore, essential to provide comprehensive information, including instrument specifications, sample preparation techniques and measurement conditions. Transparent reporting and open data practices improve the reproducibility and reliability of scientific studies.

[BEFORE_START]
Make sure following plugins are installed
- TrackMate-StarDist
- StarDist
- CSBDeep

[GUIDELINES]
N/A

[STEPS]
SECTION: Image Processing
2. Process → Noise → 2x Despecle
SECTION: Image Processing
3. Image → Adjust → Brightness/Contrast  

Move the sliders for Brightness and Contrast to the right until an evenly coloured background is created.

Brightness is set to around 80 % and Contrast to 100 %.
SECTION: Image Processing
2.1. Select "Yes" to process the whole stack. Do this with every following prompt.
SECTION: Tracking Presets
7. Now set the filters on spots.
 

SECTION: Tracking Presets
5. Plugins → Tracking → TrackMate
SECTION: Tracking Presets
6. Select StarDist detector
SECTION: Tracking Presets
4. Edit → Options → Memory & Threads → Parallel threads 1
SECTION: Tracking Presets
5.1. Specify frames (T) 
SECTION: Tracking Presets
6.1. Continue till the detection took place and keep all spots at first. 
SECTION: Tracker
10. Select LAP tracker and the parameters
 

SECTION: Tracker
8. Set the filters on tracks. 
The parameter settings must be compared with the image to remove nonsensical tracks immediately.
 

SECTION: Tracker
9. In the Display Options window, tick Display Spots and Display Tracks. It is extremely important to save the required files for further work steps. 
To do this, use the Tracks button to save the spots, edges and tracks (Export to CSV). The Spots (All Spots) can also be exported.
 

SECTION: Tracker
11. Next, take care of the TrackScheme. Here, splits, fusions, and nonsensical tracks can be removed manually.
SECTION: Expected results
12.     
 
    




SECTION: Image Processing
1. Depending on whether images of stained or unstained cells were taken, proceed as follows:
SECTION: Image Processing
2. Process → Noise → 2x Despecle
SECTION: Image Processing
3. Process → Filters → Variance → Radius 5.0 pixels
Feel free to adust the parameter to make it more suitable for your cells.
SECTION: Image Processing
4. Process → Filters → Gaussian Blur → Sigma (Radius) 2.00
Feel free to adust the parameter to make it more suitable for your cells.
SECTION: Image Processing
2.1. Select "Yes" to process the whole stack. Do this with every following prompt.
SECTION: Image Processing
5. Image → Adjust → Brightness/Contrast  

Move the sliders for Brightness and Contrast to the right until an evenly coloured background is created and the cells can be seen as blobs.

Brightness is set to around 80 % and Contrast to 100 %.
SECTION: Tracking Presets
9. Now set the filters on spots.
 

SECTION: Tracking Presets
8. Select StarDist detector
SECTION: Tracking Presets
8.1. Continue till the detection took place and keep all spots at first. 
SECTION: Tracker
10. Set the filters on tracks. 
The parameter settings must be compared with the image to remove nonsensical tracks immediately.
 

SECTION: Tracker
11. In the Display Options window, tick Display Spots and Display Tracks. It is extremely important to save the required files for further work steps. 
To do this, use the Tracks button to save the spots, edges and tracks (Export to CSV). The Spots (All Spots) can also be exported.
 

SECTION: Tracker
13. Next, take care of the TrackScheme. Here, splits, fusions, and nonsensical tracks can be removed manually.
SECTION: Expected Results
14.     
 
   
SECTION: Tracking Presets
7. Plugins → Tracking → TrackMate
SECTION: Tracking Presets
6. Edit → Options → Memory & Threads → Parallel threads 1
SECTION: Tracking Presets
7.1. Specify frames (T) 
SECTION: Tracker
12. Select LAP tracker and the parameters