Corresponding Manuscript:
"Receptor-Ligand Non-Equilibrium Kinetics (RLNEK) 1.0: An Integrated Trackmate Laminar Flow Chamber Analysis"

PyPI installation (https://pypi.org/project/rlnek):
pip install RLNEK

Anaconda installation (https://anaconda.org/zrollins/rlnek):
conda install -c zrollins rlnek

Trackmate Dependencies:
>=ImageJ 2.3.0/1.53f51
>=Java 1.8.0_301 [64-bit]
>=Trackmate v7.4.0

RLNEK Dependencies:
>= Python 3.7
>= pandas 1.3.5
>= numpy 1.22.0
>= scipy 1.7.3
>= matplotlib 3.5.1

Python (pip/conda) instructions:
1. Open terminal/command line
2. Navigate to folder with Trackmate csv files (i.e. cd /pathway/to/trackmate/csv/files)
3. Run Module script (e.g., python3 -m rlnek.Module_1)
4. Input relevant information to the RLNEK prompt (see RLNEK_tests)
5. RLNEK outputs graphs/csvs to current directory 

RLNEK Modules:
Module 0A - convert flow rate to applied tether/tensile force
Module 0B - determine non-specific bond lifetime
Module 0C - determine if coating concentration/site density range meets single-molecule criteria
Module 1 - determine Nb/NT & koff @ various flow rates, coating concentrations
NT Optimization - optimize NT values for given flow rate across various site densities (precursor to Module 2)
Module 2 - determine Nb/NT, koff, k+, & kin @ various flow rates, site densities

Tips:
1. Please read manuscript and corresponding User Guide to understand experimental parameters and data necessary to perform calculations.
2. In previous versions of Trackmate, the files are outputted in different formats. Ensure that Trackmate "tracks" and "spots" files are formatted correctly (e.g., in the "spots" file, particleIDS are listed in numerical order of trackIDs & numerical order of frames). Use provided test files as a reference.
3. All modules can handle replicates for a given condition. Please see test folders for implementation.
4. Collect Receptor-Ligand Non-Equilibrium Kinetics.
5. Use the Jupyter Notebooks versions for additional commented code, stepwise execution, and versatile parameter adjustment.
6. Trackmate user guide available in RLNEK_tests folder
7. Batch process videos with provided ImageJ script

Appendix:
Nb = number of cells/spheres bound
NT = total number of cells/spheres to cross field of view
Nb/NT = capture efficiency
koff = force-dependent dissociation rate (1/ bond lifetime)
k+ = effective on rate of cell/spheres (includes diffusive & kinetic timescales)
kin = intrinsic on rate of receptor-ligand pair (assumed constant)
***Extension of variable names/descriptions: "appendix.pdf"***

Digitized Data:
Data that was digitized from previous manuscripts and used in this RLNEK manuscript is provided in the "Digitized_Data" folder. 

Copyright Notice:
Copyright 2021-2022 Allison Chan, Zachary A Rollins, Venktesh S. Shirure, Steven C. George, and The Regents of the University of California

License Notice:
This file is part of RLNEK.

RLNEK is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.

RLNEK is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received a copy of the GNU General Public License along with RLNEK. If not, see <https://www.gnu.org/licenses/>.