Fast and flexible processing of large FRET image stacks using the FRET-IBRA toolkit.

Abstract

Ratiometric time-lapse FRET analysis requires a robust and accurate processing pipeline to eliminate bias in intensity measurements on fluorescent images before further quantitative analysis can be conducted. This level of robustness can only be achieved by supplementing automated tools with built-in flexibility for manual ad-hoc adjustments. FRET-IBRA is a modular and fully parallelized configuration file-based tool written in Python. It simplifies the FRET processing pipeline to achieve accurate, registered, and unified ratio image stacks. The flexibility of this tool to handle discontinuous image frame sequences with tailored configuration parameters further streamlines the processing of outliers and time-varying effects in the original microscopy images. FRET-IBRA offers cluster-based channel background subtraction, photobleaching correction, and ratio image construction in an all-in-one solution without the need for multiple applications, image format conversions, and/or plug-ins. The package accepts a variety of input formats and outputs TIFF image stacks along with performance measures to detect both the quality and failure of the background subtraction algorithm on a per frame basis. Furthermore, FRET-IBRA outputs images with superior signal-to-noise ratio and accuracy in comparison to existing background subtraction solutions, whilst maintaining a fast runtime. We have used the FRET-IBRA package extensively to quantify the spatial distribution of calcium ions during pollen tube growth under mechanical constraints. Benchmarks against existing tools clearly demonstrate the need for FRET-IBRA in extracting reliable insights from FRET microscopy images of dynamic physiological processes at high spatial and temporal resolution. The source code for Linux and Mac operating systems is released under the BSD license and, along with installation instructions, test images, example configuration files, and a step-by-step tutorial, is freely available at github.com/gmunglani/fret-ibra.

Plant phenotyping relevance

植物の動的な生理状態を画像から定量化するFRET画像処理ツールの開発・ベンチマークが中心であり、花粉管内カルシウム分布の抽出に実質的に応用されている。

Code and data availability

The paper's authors publicly released the FRET-IBRA analysis toolkit (Python source code, test images, example configuration files, and tutorial) under a BSD license on GitHub. The test images include the FRET microscopy image stacks of growing Arabidopsis pollen tubes used in the paper's calcium-distribution phenotypi