The problem of self-calibration of fluorescence signal in microscale sensor systems.

Most recent developments in fluorescent molecular sensor devices are based on "ON-OFF" switching, which is an operation with a single measurable parameter, commonly the quenching of total intensity. In the meantime, with this approach self-calibration on the molecular level is not achievable. This calibration is strongly needed in all kinds of microscale applications, including microarrays, microfluidic systems and living cell imaging. Different possibilities are discussed for overcoming this difficulty and an "OR-OR" switching concept is suggested that involves the two-channel detection as a promising solution. For achieving the desired efficiency specific conditions are needed: it should be a single reporter dye exhibiting rapid reversible excited-state reaction and providing two-band wavelength ratiometric response.