Background fluorescence decreases precision of fluorescence intensity measurements. (A–C) Wide-field images of 6-µm fluorescent beads, all displayed with the same scaling so relative intensity is evident. All images were collected with the same microscope (model TE2000U; Nikon) and the same camera (ORCA-AG; Hamamatsu Photonics) using a Plan-Apochromat 60x 1.4 NA oil objective lens (Nikon) and MetaMorph software. (A) Fluorescent beads (mounted in PBS) with minimal background fluorescence. A 400-ms exposure time was used, and the maximum intensity value of the beads is ∼3,800. Bar = 5 µm. (B) A solution of fluorophore (with the same spectral characteristics as the fluorophore in the bead, diluted in PBS) was added to the specimen to increase the background fluorescence. The exposure time had to be decreased to 100 ms to get the same maximum intensity value of the beads, ∼3,800. (C) Image B, after background subtraction. Because a shorter exposure time was used in B, fewer photons from the beads were collected than in A. Collecting fewer photons from the object of interest means a higher contribution of Poisson noise, and less precise quantitation of fluorescence intensity values. Therefore, one should work to remove background fluorescence from the image (see Table I) before background subtraction.