Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

Abstract

Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the threedimensional compartmentalization of synaptic vesicle trafficking in live human neurons. ResultsDesign, synthesis, and mechanistic studies. To create photoregulated fluxional fluorophore PFF-1 ( Fig. 2a), we grafted an acylhydrazone photoswitchable unit 30,31 onto a rhodamine B scaffold. Probe PFF-1 was synthesized in two steps and high yields from rhodamine B (Fig. 2b). As a mechanistic control compound, we also synthesized probe 2, which could be obtained from intermediate 3 (Fig. 2b). We hypothesized that prior to photoactivation, PFF-1 should exist predominantly as the E isomer of the acylhydrazone and a dark, non-fluxional, spirocyclized derivative ( Fig. 2a). This structure was confirmed by X-ray crystallography ( Supplementary Fig. 1) and electronic absorption spectroscopy ( Supplementary Fig. 2). Upon photoisomerization with light of 410 nm, the fluorescence of a solution of PFF-1 in aqueous buffer (pH = 7) increases by 12-fold, whereas photoirradiation of compound 2 gives only a 1.6-fold increase under the same conditions (Fig. 2c). Furthermore, photoactivation of PFF-1 at pH = 5 led to an even larger increase in fluorescence of 22-fold, but for compound 2 the increase was only 3-fold. 1 H NMR and HPLC analysis of PFF-1 prior to photoactivation confirmed that, in solution, virtually all molecules are present as the E isomer ( Supplementary Fig. 3).Upon irradiation in a UV photoreactor (10 min, 375 nm) a photostationary state was reached with about 17% conversion of the E isomer into the Z form, as determined by both 1 H NMR and HPLC analysis ( Supplementary Fig. 3). Photoisomerization using an LED source (410 nm, 8.5 mW cm -1 ) proceeded with a quantum yield of 0.4(2)% at pH = 5 and 0.5(5)% at pH = 7.4 ( Supplementary Figs. 4-6), confirming that the photochemical process does not depend strongly on pH. After photoirradiation, the Z isomer did not revert to the E form thermally over a period of 2 h ( Supplementary Fig. 7). 10 ms). After this initial photoactivation step, cells were either imaged using only the fluxionality of PFF-1 (single photoactivation, Fig. 4a,c) or by repeating the photoactivation pulse every 10 min to replenish the population of fluxional molecules (sequential photoactivation, Fig. 4b,c...