The concept of λ-ratiometry in fluorescence sensing and imaging.

Very limited number of parameters is available for fluorescence sensing and imaging. The changes of intensity are of low analytical value due to the absence of internal reference. Anisotropy and lifetime sensing have their own limitations. In this respect the λ-ratiometric (based on intensity ratios at two or more wavelengths) recording of spectral changes becomes more popular. Because the spectral changes are connected directly with the variations of interaction energies this approach is seen as the most universal method to study intermolecular interactions. It is applicable for different sensor formats and for obtaining analytical information from cell images. Here we critically analyze different approaches in λ-ratiometric sensing that use single and double fluorescence emitters and are based on different mechanisms producing spectroscopic change. Very promising is the exploration of mechanisms that allow obtaining ratiometric response from a single dye.