Installation instructions:

You can install the development version from GitHub with:

# install.packages("devtools")
devtools::install_github("dmattek/ARCOS")

Package Specific Documentation and usage instructions:

Github repository:

How to get the necessary input data for ARCOS

To get from raw data to data that ARCOS accepts as input, several different tools can be used. The steps neccessary can be summarised as follows:

1. Acquire your image data.

2. Segment images to identify individual objects.

   • Tools that can be used for this purpose include Ilastik (a more general approach), simple image thresholding, or more specialised tools such as stardist, and cellpose in the case of microscopy data and many others.

3. Optionally, extract a measurement value from objects, for example from a fluorescent biosensor, other intensity changes, shape changes or something else entirely. This is needed if some objects should be excluded from the analysis based on a activity threshold.

4. Optionally, track detected objects across time and space. This is needed from subsequent downstream analysis but not necessarily for ARCOS itself.

How to prepare the data with ARCOS

Interpolate Measurments

If the measurement column contains missing values, in the first step the data should be interpolated. Helper functions are available across all implementations.

Detrend and rescale the measurement

Often time-series data can exhibit local and global trends that are introduced as artefacts of the data acquisition. In the case of fluorescence microscopy bleaching can be an example of such a bias. To help with this several de-trending methods are implemented in ARCOS, all of which are optional. There are three options available: ['none', 'lm', 'runmed']. With "none", the data is first rescaled to a feature range of 0,1. Subsequently, a short-term median filter is applied to the time series to reduce noise in the data. "lm" uses linear regression to detect long-term trends. Additionally, a short-term median filter is used to reduce noise in the time series. With "runmed", a long-term and a short-term median filter is used. After de-trending the data with options "lm" and "runmed", the output is rescaled to a feature range of 0,1.

Binarization

ARCOS requires binarized data to detect and track collective event clusters. Binarization is done by setting a threshold and defining measurements below this threshold as 0 and above as 1. Alternatively, all input objects can be defined as "active" if no measurement is available.

We provide several implementations of ARCOS as open-source code which can be found on github.

The code provided as R and Python packages can be used in Jupyter notebooks or in batch processing pipelines. The Napari plugin enables anyone without extensive programming knowledge to explore parameters through an intuitive GUI on a platform that emerges as a de-facto standard for viewing multidimensional images

About

ARCOS is a computational method to detect and quantify collective, spatio-temporally correlated phenomena. The algorithm identifies and tracks spatial clusters in time-lapse images. Although designed to analyze signalling phenomena in biological cells or cell collectives, it is applicable to other systems even outside of the realm of cell biology.

Code

We provide open-source implementations of ARCOS for:

Both code bases can handle segmented data and raster images, although the Python version contains additional optimizations and features to handle large datasets.

GUI

Additionally, two dedicated interactive plugins for are available:

• − to handle data from image segmentation

• − to handle raster images

The napari plugins enables anyone without extensive programming knowledge to explore parameters through an intuitive GUI on a platform that emerges as a de-facto standard for viewing multidimensional images.

Demos

• Demo of the arcos-gui napari plugin ()

• Demo of the entire workflow from raw images to detection of collective events ()

Publications

The original ARCOS algorithm is now published in the Journal of Cell Biology (JCB):

For a complete tutorial on how to use ARCOS from raw images to full analysis refer to our publication in Methods in Microscopy (MiM):

and the corresponding containing a detailed notebook and installation instructions.

If you use this method in your research, please cite our papers:

and if you used the tutorial please also cite:

How to use ARCOS

How ARCOS works

Learn the fundamentals of ARCOS to get a deeper understanding of our main features:

Arcos is aimed at analysing time series data which should be arranged in long format. Each row should define the object’s location, time, and optionally the measurement value. For the implementations in R and python, the objects don't necessarily need to be tracked over time. However, it is recommended as it allows a more in-depth downstream analysis of ARCOS's output.

An example dataset could look like this:

For the napari-plugin, as of now, a track id is required.

Identification of spatial clusters

A dbscan algorithm first spatially clusters entities deemed active in each frame. In step 2, clusters are sequentially linked between frames to capture collective events over time. The cluster in frame one forms a seed of a collective event. The cluster in frame two is linked to this seed cluster because several of its member cells are within the neighbourhood radius 𝜀. In frame three, only clusters #3, #5, & #6 are linked to the previous frame’s cluster. Cells in cluster #4 are too far and thus form a new seed of a collective event.

Flow-Chart overview of ARCOS event detection

The flow chart below outlines the steps in the algorithm.

Napari

is a multidimensional image viewer for python. It is available either as a python package or as a standalone application. See for installation instructions of napari.

The following describes an example image processing pipeline written in python to analyse collective events in an MDCK epithelium. What we are aiming for can be seen in the gif above. First, the erk measurement is extracted from the images and the individual nuclei are tracked. The second step shows how to analyse the data with ARCOS. Similar results can be achieved with standalone tools such as CellProfiler and Ilastik. And both the R and Python packages can be used after preparing the data.

In the following zip file, you can find both the input data and the generated output from the image segmentation pipeline and ARCOS.

Data Preparation

The data in this example is from an MDCK cell line that stably expresses a fluorescent FRET biosensor reporting the activity of the ERK kinase.

In the first step, the nuclei are segmented using the stardist python package.

Subsequently, the mean intensity of individual objects in the ratio image is measured and the individual nuclei are tracked over time.

Import libraries and define custom functions

Set variables

Load image and stardist model

Segment Nuclei and extract values from image

Track Nuclei and save csv

Analyse data with ARCOS

In this example, the python package arcos4py is used. But the same analysis could be carried out easily with the R package or the napari plugin.

Imports

Detect Collective Events

Filter Collective Events

Plot NoodlePlot

Visualize Events in Napari

This step is optional and only one way to visualize events but can be usefull to validate correct event detection.

Imports

Prepare Data

Remap measured Ratio to segmented labels

Open Napari and add Layers

Installation instructions:

To install from pip, run this command in your terminal:

Installing from conda-forge can be achieved by adding conda-forge to your channels with:

Once the conda-forge channel has been enabled, arcos4py can be installed with the conda package manager: