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.
Highlights
IntroductionIn most common microscopy configurations, including those used in fluorescence microscopy, the light source is aligned for Koehler illumination [1], which ensures that the structure of the light source (as either rectangles generated by the LEDs or a light bulb’s coil) does not introduce optical artefacts in the microscopy pictures
In most common microscopy configurations, including those used in fluorescence microscopy, the light source is aligned for Koehler illumination [1], which ensures that the structure of the light source does not introduce optical artefacts in the microscopy pictures
This table is to guide the user through the steps needed to generate a matrix that represents the nonuniform illumination in a sample picture, along with examples in both MATLAB and python to arrive at the same result
Summary
In most common microscopy configurations, including those used in fluorescence microscopy, the light source is aligned for Koehler illumination [1], which ensures that the structure of the light source (as either rectangles generated by the LEDs or a light bulb’s coil) does not introduce optical artefacts in the microscopy pictures. Such illumination provides a clean light with a radial symmetric Gaussian intensity gradient and a non-uniform illumination. The image depict samples of high optical density [Fig. 4] or a microfluidic device [10] (originally designed to study the diffusion of fluorophores and single cell motility in laminar flows [10]), [Figs. 2 and 3]
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