Matrix-masking to balance nonuniform illumination in microscopy

Research output: Contribution to journalArticle

Abstract

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.

Details

Authors
Organisations
External organisations
  • University of Glasgow
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medical Image Processing
Original languageEnglish
Pages (from-to)17279-17288
Number of pages10
JournalOptics Express
Volume26
Issue number13
Publication statusPublished - 2018 Jun 25
Publication categoryResearch
Peer-reviewedYes