Solvatochromic Polarity-Sensitive Probes for Live Cell Imaging

Abstract

Polarity-dependent fluorescent probes are attracting increasing interest for high-resolution cell imaging. A notable example is that of solvatochromic dyes located in a domain where polarity changes occurring upon binding lead to the enhancement of the fluorescence signal. This can yield a very sensitive detection of molecular recognition events that is applicable even in the case of non-overexpressed proteins.1 We shall present a toolbox of new solvatochromic coumarin derivatives that are suitable for in vivo imaging applications.Following a preliminary screening by computational methods of a set of candidate-structures selected on the basis of their spectral properties, we designed a synthetic protocol allowing a broad range of substitution patterns. We shall present experimental data on several probes showing excellent fluorescence quantum yields (up to 0.95), high molar-extinction coefficients (up to 46,000 M−1cm−1), and large Stokes shifts. Notably, our molecules display marked solvatochromism: they are virtually non emissive in water, but intensely fluorescent in less polar media (we shall show up to 780-fold fluorescence enhancement).We shall also report on probe suitability for in vivo experiments. When tested on cultured cells, these coumarins proved not harmful and their photophysical properties were unchanged compared to data in solution. Owing to both their strong solvatochromic properties and their lipophilic character, the coumarins fluoresce only in the most lipophilic environments of the cell. In particular, colocalization experiments with organelle markers indicate that our coumarins locate in ER, membranes and lysosomes, depending on their chemical structure. Finally, one compound shows monoexponential decay of fluorescence with a lifetime which is linearly dependent on solvent polarity. We shall discuss how this feature promotes its use as ratiometric indicator of cell polarity at the nanoscale level.1) Nalbant, P.; Hodgson, L.; Kraynov, V.; Toutchkine, A.; Hahn, K. M. Science2004, 305, 1615-1619.