Luminescence assay using cyclical excitation wavelength sequence

A method of effecting measurement of proximity between luminescent species based on detection of transfer of excitation energy therebetween, the method comprising providing a first photoluminescent species (the ‘donor’) and a second photoluminescent species (the ‘acceptor’) which are such that the donor species and the acceptor species have at least some excitation spectral regions which differ and also such that at least a part of the emission spectrum of the donor overlaps with at least a part of the excitation spectrum of the acceptor,

• exciting the donor species with an excitation source, and
• analyzing emission of the acceptor using a detection means,
• characterized in that the donor species is excited with a cyclical temporal sequence of wavelength bands, optionally provided as pulses or modulated in intensity, giving rise to a characteristic temporal fluctuation in emission from the donor species, and
• emission in at least one wavelength band characteristic of the acceptor is analyzed to detect the presence of the said characteristic fluctuation or a subcomponent thereof and optionally also to detect a fluctuation characteristic of direct excitation of the acceptor.