Table 2. Useful known samples for imaging system validation.

Sample	Error	References for correction protocols
(a) Fluorescent microspheres (beads) that are below the diffraction resolution limit of the imaging system	Optical aberrations	Hiraoka et al., 1990; Goodwin, 2013
(b and c) Multi-wavelength beads below the diffraction resolution limit of the imaging system	Channel registration	Hibbs et al., 2006; Spiering et al., 2013; Wolf et al., 2013
(d) Stage micrometer	In magnification/pixel size	Wolf et al., 2013
Flatfield slide	Nonuniform illumination	Model, 2006
Single-labeled biological sample	Bleed-through	Spiering et al., 2013
		Bolte and Cordelières, 2006
Stable biological sample	Photobleaching	Bancaud et al., 2010
Unlabeled biological sample	Autofluorescence	Hibbs et al., 2006

We used (a) Molecular Probes FluoSpheres; (b) for high-resolution imaging, Invitrogen TetraSpeck Microspheres, 0.1 µm; (c) for low-resolution imaging, Invitrogen FocalCheck Beads, 6 µm or 15 µm; and (d) MicroScope World, 25 mm KR812.