FLINO-A new method for immunofluorescence bioimage normalization

John Graf, Sanghee Cho, Elizabeth McDonough, Alex Corwin, Anup Sood, Andreas Lindner, Manuela Salvucci, Xanthi Stachtea, Sandra Van Schaeybroeck, Philip D Dunne, Pierre Laurent-Puig, Daniel Longley, Jochen H M Prehn, Fiona Ginty

Research output: Contribution to journalArticlepeer-review

5 Downloads (Pure)


Multiplexed immunofluorescence bioimaging of single-cells and their spatial organization in tissue holds great promise to the development of future precision diagnostics and therapeutics. Current multiplexing pipelines typically involve multiple rounds of immunofluorescence staining across multiple tissue slides. This introduces experimental batch effects that can hide underlying biological signal. It is important to have robust algorithms that can correct for the batch effects while not introducing biases into the data. Performance of data normalization methods can vary among different assay pipelines. To evaluate differences, it is critical to have a ground truth dataset that is representative of the assay. A new immunoFLuorescence Image NOrmalization (FLINO) method is presented and evaluated against alternative methods and workflows. Multi-round immunofluorescence staining of the same tissue with the nuclear dye DAPI was used to represent virtual slides and a ground truth. DAPI was re-stained on a given tissue slide producing multiple images of the same underlying structure but undergoing multiple representative tissue handling steps. This ground truth dataset was used to evaluate and compare multiple normalization methods including median, quantile, smooth quantile, median ratio normalization (MRN) and trimmed mean of the M-values (TMM). These methods were applied in both an unbiased grid object and segmented cell object workflow to 24 multiplexed biomarkers. An upper quartile normalization of grid objects in log space was found to obtain almost equivalent performance to directly normalizing segmented cell objects by the middle quantile. The developed grid-based technique was then applied with on-slide controls for evaluation. Using five or fewer controls per slide can introduce biases into the data. Ten or more on-slide controls were able to robustly correct for batch effects. Supplementary data are available at Bioinformatics online.
Original languageEnglish
JournalBioinformatics (Oxford, England)
Early online date02 Oct 2021
Publication statusEarly online date - 02 Oct 2021

Bibliographical note

© The Author(s) 2021. Published by Oxford University Press.


Dive into the research topics of 'FLINO-A new method for immunofluorescence bioimage normalization'. Together they form a unique fingerprint.

Cite this