Matrix-masking to balance nonuniform illumination in microscopy

Nordenfelt, P., Cooper, J. M. and Hochstetter, A. (2018) Matrix-masking to balance nonuniform illumination in microscopy. Optics Express, 26(13), pp. 17279-17288. (doi: 10.1364/OE.26.017279)

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With a perfectly uniform illumination, the amount and concentration of fluorophores in any (biological) sample can be read directly from fluorescence micrographs. However, non-uniform illumination in optical micrographs is a common, yet avoidable artefact, often caused by the setup of the microscope, or by inherent properties caused by the nature of the sample. In this paper, we demonstrate simple matrix-based methods using the common computing environments MATLAB and Python to correct nonuniform illumination, using either a background image or extracting illumination information directly from the sample image, together with subsequent image processing. We compare the processes, algorithms, and results obtained from both MATLAB (commercially available) and Python (freeware). Additionally, we validate our method by evaluating commonly used alternative approaches, demonstrating that the best nonuniform illumination correction can be achieved when a separate background image is available.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Hochstetter, Dr Axel and Cooper, Professor Jonathan
Authors: Nordenfelt, P., Cooper, J. M., and Hochstetter, A.
College/School:College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:Optics Express
Publisher:Optical Society of America
ISSN (Online):1094-4087
Published Online:20 June 2018
Copyright Holders:Copyright © 2018 Optical Society of America
First Published:First published in Optics Express 26(13): 17279-17288
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
617021Advanced Diagnostics using PhononicsJonathan CooperEngineering and Physical Sciences Research Council (EPSRC)EP/K027611/1ENG - BIOMEDICAL ENGINEERING