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A fluorescent microsphere is a small spherical particle containing or carrying fluorescent material. Excitation light produces a detectable emission signal, allowing the particle to serve as a tracer, positional marker or calibration object.

For imaging resolution measurements, the bead should be small relative to the instrument's point spread function; otherwise the recorded image includes the bead's finite size as well as the optical blur. In flow experiments, its motion tracks the fluid only when buoyancy, inertia and surface interactions do not materially bias the trajectory. The same bright signal can therefore support different measurements with different assumptions.

A researcher whose team investigated how fluorescent microsphere size affects measurements of a confocal point spread function. A finite bead is an extended object, so its image includes both optical blur and its own dimensions. The study combines numerical calculations with measurements to assess that calibration bias.^[ref]

A researcher whose team examined the use of fluorescent microspheres as optical calibration objects. Their confocal-microscopy study shows how the marker’s finite size changes the measured point spread function, linking the physical test object with the apparent resolving performance of the instrument.^[ref]