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

If you use functions in the notebooksLoading and preparing imagesorQuantification of images please cite:Liu et al. (2017)Low-abundant species facilitates specific spatial organisation thatpromotes multispecies biofilm formation.Envir.Microbiol.

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

If you use theclumps oroccupancy 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 allanalyses

Quantification ofimages

Co-aggregation and relativepositioning

Dynamic sectioning of images inz-direction

Detectingaggregates

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

loadIMG Load .tif files and turn them into arrays, and save them asRDS 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 channelat each layer.

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

layer_split Splits quantification in Top, Middle and Bottom based onfill 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. Anundirected version ofoccupancy

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

create_random Create random images for testing

create_nulls Create image files for the empty spaces in an image, suchthat this can be used in the analysis. For example, it can then becalculated how much empty space there is a around a certain channel withoccupancy

extract_layers With the output fromlayer_split it makes a list ofwhat layers to analyze inoccupancy,clumps,co_agg andcross_ratio

xy_splits Splits the image in each xy position and then run ananalysis

clumps Detects and quantifys clumps by aggregating pixels

clumps_plot Plots the output fromclumps

An internal function,tiff_to_array, is partly borrowed fromhttps://github.com/rmnppt/iMage. Furthermore, some of the algorithmicframework for theco_agg,occupancy andcross_ratio analysis isalso borrowed from this repository.