iTACS overview

For a detailed protocol, please refer to Nguyen et. al., JoVE 181, e63095 (2022)

Need guidance to implement iTACS on your microscope or looking for collaboration?
We will be glad to hear from you: dtambe@southalabama.edu

Here is an overview of an iTACS-based experiment to examine physiological properties of individual adherent cells:

Installing Analysis and Visualization Module (AnViM)

Current version is developed and tested on Ubuntu operating system (18.04 - 20.10). One of the best ways to use the software on other operating system is through virtual Ubuntu machine which involves installing Oracle VM VirtualBox and on it install Ubuntu.

Task	Links
Install gfortran using sudo apt install gfortran
In the root folder, create a directory /opt/
Install Fiji in /opt/Fiji.app/	https://imagej.net/software/fiji/downloads
Save a copy of Radial_Profile.java in the plugins folder of Fiji	https://imagej.nih.gov/ij/plugins/radial-profile.html
Install pluginsImageJ-MATLAB, BIG-EPFL, and BioVoxxel using Help > Update > Manage update sites from Fiji	https://imagej.net/update-sites/setup
Install plugin iterativePIV by adding the link https://sites.imagej.net/iterativePIV/ to Help > Update > Manage update sites > Add update site	https://imagej.net/update-sites/setup
Place the files edge, inti, island, and strip in the folder /opt/MSM/	https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AnViM/opt/iTACS/MSM/edge https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AnViM/opt/iTACS/MSM/inti https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AnViM/opt/iTACS/MSM/island https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AnViM/opt/iTACS/MSM/strip

Software to compute cell-ECM forces

• Process used to compute cell-ECM forces is not included in this package, but any software that takes in the hydrogel surface deformation as input and provides forces exerted by a sheet of cells as output will work. Here is a software that will work for a thick hydrogel: FTTC.
• The cell-ECM forces are then used by the inti, edge, and strip software to quantify mechanical stresses within the cellular cluster. These software expect the cell-ECM forces to be in analysis/p*/traction/*_trac.dat (where the first * is position number [0, 1, 2, 3, ...] and the second * is the file number [0001, 0002, 0003, ...]) files with four columns: x y Tx Ty. Unit of x and y: pixel and Tx and Ty: Pascal.

Installing Acquisition and Training Module (AcTrM)

AcTrM is developed and tested on Micro-Manager-2.0beta on Windows 10.

Task	Links
Install Micro-Manager 2.0 beta	https://valelab4.ucsf.edu/~MM/nightlyBuilds/2.0.0-beta/Windows
Establish a connection between Micro-Manager software and the microscope hardware	https://micro-manager.org/Device_Support https://micro-manager.org/Micro-Manager_Configuration_Guide
Save a copy of Turboreg_.jar in the plugins folder of Micro-Manager	http://bigwww.epfl.ch/thevenaz/turboreg/
In the plugins folder of Micro-Manager, create a subfolder IMBL and copy in this subfolder the files AcTrM.bsh, AcTrM_functions.bsh, and ColumnComparator.java	https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AcTrM/Micro-Manager-2.0beta/plugins/IMBL/AcTrM.bsh https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AcTrM/Micro-Manager-2.0beta/plugins/IMBL/AcTrM_functions.bsh https://github.com/IntegrativeMechanobiologyLaboratory/iTACS/blob/main/AcTrM/Micro-Manager-2.0beta/plugins/IMBL/ColumnComparator.java

Analyzing the demo files using AnViM

• Create a folder tnimgs in a suitable directory. Ensure that the path of this directory does not have space in the names of any of the parent folders.
• Copy all the c*_p*_t*.tif files from GitHub into the tnimgs folder.
• From Fiji, use MSM > MSM - pre-processing to start data analysis and follow the procedure described in the Nguyen, Battle, Paudel et al., manuscript.
  

How to cite iTACS

Nguyen, A., Battle, K.C., Paudel S.S., et al. Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence. J. Vis. Exp. 181, e63095 (2022).
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Accompanying JoVE Video

Notes

To segment smooth muscle cells that are separated and have long and narrow extensions,

• conduct cluster segmentation by thresholding to have white cells (pixel value 255) and black background (pixel value 0).
• for individual segmentation, start with parameter values: sledgehammer = 0, proninance = 100, mallet = 40