BPS2026 – Characterization of a novel phosphorous rhodamine dye for optical voltage sensing

NIR-absorbing phosphorous-substituted rhodamine dyes were shown to have outstanding properties as labeling agents in long-term and deep tissue imaging due to their high chemical and photochemical stability, surpassing that of their oxygen- and silicon-substituted counterparts. [1–3] Here, we report and characterize a novel phosphorous rhodamine dye, JMS-13-01, as a tool compound for optical voltage sensing. By introduction of a phenyl group at the phosphorous atom we aim to achieve improved optical properties and biological responses over the known methyl group derivatives [4] especially focusing on fast responses. The photophysical characterization of the dye in terms of excitation and emission maxima, absorption coefficient ε, fluorescence quantum yield ϕ F , and fluorescence lifetime τ, is reported in organic media (λ ex = 690 nm, λ em = 730 nm, ε max = 81,500 M −1 cm −1 , ϕ F = 0.02, τ = 2.70 ± 0.09 ns) as well as in a buffer system (λ ex = 715 nm, λ em = 744 nm, ε max = 80,500 M −1 cm −1 , ϕ F = 0.02, τ = 1.65 ± 0.01 ns) for the intended biological applications. The dye voltage-reporting properties were evaluated on Xenopus laevis oocytes in a cut-open oocyte vaseline gap (COVG) voltage-clamp apparatus with epifluorescence capability (voltage-clamp flurometry). The dye showed near-linear deflections of the same sign as the voltage change over ±200 mV, with amplitude (ΔF/F) 2.1 ± 0.39% between −100 and 0 mV (a minimal estimate, as autofluorescence was not subtracted). In a representative pulse (−80 to +200 mV), the depolarization was reported with time-constant τ = 88.5 μs and the repolarization with τ = 56.6 μs (clamp τ was 10 μs). Under action-potential (AP) clamp, the dye faithfully reported a human ventricular AP, including its phase-1 notch, and a series of DRG small-neuron APs, with modest photobleaching under continuous-wave excitation. [1] Grzybowski, M. et al., (2018). Angew. Chem . Int. Ed. 57, 10137–10141. [2] Grimm, J.B. et al., (2020). Nat. Methods , 17, 815–821. [3] Ogasawara, H. et al., (2018). Chem. Commun ., 54, 299–302. [4] Gonzales, M. et al., (2021). J. Am. Chem. Soc. , 143(5), 2304–2314.