WG 3 – Uniformity of field – flatness

Illumination field uniformity is critical for quantitative imaging when comparing fluorescence intensities across a field-of-view (FOV) or a large tile of images capturing an entire sample. If the illumination is not constant over a large area, the fluorescence intensities will not represent the inherent fluorescence but rather the location within the image. Thus, WG3 aims at defining a set of universal protocols to assess the uniformity of illumination (i.e., “flatness” of field) over the FOV of any photon-based imaging system and allow for correction of any non-uniformity. These protocols will identify the necessary tools, the procedures required to perform the measurements, and the analysis methods required for their interpretation. WG3 will also define criteria regarding the cut-off for acceptability and the need for correction. A database will be created with ideal images of uniform fields-of-view from different microscope modalities and settings to be used as a reference by the community and validate the protocol and criteria.