Abstract
This paper discloses the design, synthesis, and imaging applications of the first member of a new class of SPOTs, small-molecule photoactivatable optical sensors of transmembrane potential. SPOT2.1.Cl features an established voltage-sensitive dye, VoltageFluor2.1.Cl--or VF--capped with a dimethoxy-o-nitrobenzyl (DMNB) caging group to effectively diminish fluorescence of the VF dye prior to uncaging. SPOT2.1.Cl localizes to cell membranes and displays weak fluorescence until photoactivated. Illumination generates the parent VF dye which then optically reports on changes in the membrane voltage. After photoactivation with spatially restricted light, SPOT2.1.Cl-loaded cells display bright, voltage-sensitive fluorescence associated with the plasma membrane, while neighboring cells remain dark. Activated SPOT reports on action potentials in single trials. SPOT can be activated in neuron cell bodies or uncaged in dendrites to enable structural tracing via "backfilling" of the dye to the soma, followed by functional imaging in the labeled cell. The combination of cellular specificity achieved through spatially defined patterns of illumination, coupled with the fast, sensitive, and noncapacitive voltage sensing characteristics of VF dyes makes SPOT2.1.Cl a useful tool for interrogating both structure and function of neuronal systems.