Why Illumination Uniformity Matters

Reliable and accurate quantitative comparisons of fluorescence intensities across an image are impossible when the field illumination profile is not uniform. Uneven illumination of the sample will cause the intensity of a feature in one region of the field of view to register a different magnitude than the intensity of a feature of equal fluorophore concentration in another region of the field of view. When trying to determine the level of expression of a fluorescently tagged protein within a population of cells with uneven illumination, cells in the center of the image will appear to be brighter than those at the edges of the image.

This problem can lead to incorrect interpretation and analysis of the image data, especially when comparisons are made between images captured in different spectral channels (ie: FRET, calcium ratio imaging, colocalization, etc.).