Latest article: analyzing fluorescence
Fluorescence is a powerful and versatile method that is widely used in several scientific fields. However, when it comes to analyze the origin of a given fluorescence signal (i.e. calibrating the excitation intensity, the number of emitters and their respective quantum yield), fluorescence spectroscopy is intrinsically limited, as it is unable to separately quantify the excitation intensity from the number of fluorescent emitters. This may cause severe misinterpretation of experimental results as soon as complex systems are used, such as plasmonic metal substrates or scattering media. The range of application is large, from bioassays to microscopy imaging.
We address this issue in a recent Optics Letters publication by monitoring higher-order harmonic fluorescence signals upon harmonic excitation modulation. To our knowledge, this is the very first method able to quantify the excitation intensity and the number of emitters separately. It is a significant supplement to the fluorescence toolbox. The method is compatible with a wide range of observations, and relatively simple to implement. This opens new characterization routes for applications on surface-enhanced fluorescence bioassays, microscopy across scattering samples, and deep tissue fluorescence imaging.