Engineering a FRET strategy to achieve a ratiometric two-photon fluorescence response with a large emission shift and its application to fluorescence imaging

the high degree of overlap of two emissions bands and shifts of the TPE maxima. To achieve the desired TPE-based sensing and to circumvent the problems stated above, we adapted a Förster resonance energy transfer (FRET) strategy to develop small molecule ratiometric TPE fluorescent probes. Our FRET-based ratiometric TPE fluorescent probe displays a remarkable emission shift (up to 125 nm) with two well-resolved emission bands. Hence the ratio of these two emission bands could enable the measurement of fluorescence changes more accurately, thus further improving imaging in live cells and deep tissues. To the best of our knowledge, the current reported probe has the largest emission shift among all the small molecule ratiometric TPE fluorescent probes while the maximum TPE wavelength remains unchanged. This work has provided a FRET approach to fabricate novel small molecule ratiometric TPE fluorescent probes that improve imaging in deep tissues.