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The uniformity of field is a well-known parameter related to the inhomogeneous spatial distribution of the intensity in the image of an homogeneous (and flat) specimen.
In any fluorescence microscope, knowledge of the uniformity of field is important when measuring image intensity. For intensity quantification in images of biological samples, the uniformity of field must be known, and eventually corrected, to ensure accurate measurements.
The “uniformity of field” analysis provides the spatial distribution of the intensity, due to illumination and detection inhomogeneities, within the field of view.
The “uniformity of field” analysis is associated with any “homogeneous specimen” (see Figure 1). A “homogeneous specimen” can be a thin uniform fluorescent layer, a concentrated fluorescent dye with strong absorption at the excitation wavelength, a bulk fluorescent material, or a fluorescent liquid.
Figure 1: Example image of a “homogeneous specimen”, fulfilling the acquisition recommendations.