This is an R package for various 3D analyses on confocal images of microbial biofilms, microcolonies and communities.

If you use functions in the notebooks Loading and preparing images or Quantification of images please cite: Liu et al. (2017) Low-abundant species facilitates specific spatial organisation that promotes multispecies biofilm formation. Envir. Microbiol.

If you use the co_agg function please cite: Liu et al. (2018) Micro-scale intermixing; a requisite for stable and synergistic co-establishment in a four-species biofilm. ISMEJ

If you use the clumps or occupancy functions please cite: Røder et al. (2018) Enhanced bacterial mutualism through an evolved biofilm phenotype. ISMEJ

# devtools has to be installed (install.packages("devtools"))
devtools::install_github("Russel88/RCon3D")

Loading and preparing images, required for all analyses

Quantification of images

Co-aggregation and relative positioning

Dynamic sectioning of images in z-direction

Detecting aggregates

Most functions can run in parallel by setting the cores argument. This will highly speed up analyses, but will also use more memory.

loadIMG Load .tif files and turn them into arrays, and save them as RDS files ready for downstream analysis

findIMG Find already loaded images in a set directory

smoothIMG Median smooth images

morphIMG Apply mathematical morphology kernel on image

merge_channels Create arrays based on combinations of channels. E.g. intersects, unions or subtractions

quant Quantifies the number of pixels in each image, for each channel at each layer.

layer_stand Standardize layers based on fill. Relevant if the bottom of the specimen is the layer with highest fill

layer_split Splits quantification in Top, Middle and Bottom based on fill and/or a set number of layers

occupancy Estimates the proportion a (target) channel occupy around a (focal) channel

co_agg Estimates the co-aggregation between two channels. An undirected version of occupancy

cross_ratio Estimates the ratio between two channels (targets), at some distance from a focal channel

create_random Create random images for testing

create_nulls Create image files for the empty spaces in an image, such that this can be used in the analysis. For example, it can then be calculated how much empty space there is a around a certain channel with occupancy

extract_layers With the output from layer_split it makes a list of what layers to analyze in occupancy, clumps, co_agg and cross_ratio

xy_splits Splits the image in each xy position and then run an analysis

clumps Detects and quantifys clumps by aggregating pixels

clumps_plot Plots the output from clumps

An internal function, tiff_to_array, is partly borrowed from https://github.com/rmnppt/iMage. Furthermore, some of the algorithmic framework for the co_agg,occupancy and cross_ratio analysis is also borrowed from this repository.