Photophysical properties of Na⁺ -indicator dyes suitable for quantitative two-photon fluorescence-lifetime measurements

Two-photon microscopy (2PM) offers great potential in fluorescence imaging of intracellular Na⁺ dynamics of live cells. A severe drawback, however, is that quantitative ratioing of fluorescence intensities at different wavelengths [possible in one-photon imaging with the classical Na⁺ -indicator dye sodium-binding benzofuran isophtalate (SBFI)] is not practical in 2PM. We aimed at establishing 2PM-based time-correlated fluorescence lifetime measurements as an alternative method for quantifying Na⁺ dynamics. We compared the photophysical properties of the four Na⁺ -sensitive fluorescent indicator dyes SBFI, CoroNa Green, Sodium Green and Asante NaTRIUM Green-2 (ANG-2) in cuvette calibrations. All four dyes showed Na⁺ -dependent intensity changes, with ANG-2 having the most favourable properties for 2PM. All dyes but SBFI showed significant changes in their fluorescence lifetime upon Na⁺ binding, again with ANG-2 being the most promising dye. We found that, unfortunately, the fluorescence lifetime of ANG-2 is not only affected by Na⁺ but also by protons, K⁺ and dye impurities, rendering a quantitative description of the individual lifetime components impractical. However, a simplified calibration procedure, based on a published approach for Ca²⁺ imaging, allowed relating lifetimes to Na⁺ concentration. Using ANG-2 and the simplified calibration will allow quantitative two-photon Na⁺ imaging with millimolar sensitivity.

Lay description: Dynamic changes of ion concentrations, which play crucial roles in cellular physiology, can be monitored with appropriate fluorescent indicator dyes. For intracellular sodium ions (Na⁺ ), certain dyes even allow quantitative measurements with standard microscopic techniques. However, for two-photon microscopy, which allows resolving cells deep in intact tissue, imaging solutions that are fully quantitative are lacking. For the four commercially available Na⁺ dyes 'SBFI', 'CoroNa Green', 'Sodium Green', and 'Asante NaTRIUM Green-2' (ANG2) we analyzed whether their fluorescent lifetime (LT), i.e., the nanosecond decay of emission of photons after a pulsed excitation, could serve as a quantitative measure of intracellular Na⁺ . Pulsed excitation in the femtosecond range is an inherent feature of two-photon microscopy and, in combination with fast, single-photon counting microscopes, allows for easy-to-implement LT microscopy. We found that Sodium Green and ANG2 showed strong Na⁺ -dependent changes in the fluorescence LT, while SBFI showed no, and CoroNa Green only small changes. ANG2, as the brightest dye, was further characterized regarding effects of protons and potassium ions (K⁺ ), both also present in cells at significant concentrations, on the fluorescence LT. We found that the LT of ANG2 is affected in a predictable manner by Na⁺ , K⁺ , and protons. However, our data reveal that the commercial dye must also contain impurities with unexpected Na⁺ - and K⁺ -binding characteristics, rendering a quantitative description of the individual lifetime components impractical. We, therefore, adapted a simplified calibration procedure, based on a published approach for Ca²⁺ imaging, that allows relating the average lifetime to Na⁺ concentration. With this simplified calibration procedure, ANG2 is well suited for quantitative two-photon Na⁺ imaging with millimolar sensitivity.