A Universal Photoactivatable Tag Attached to Fluorophores Enables Their Use for Single‐Molecule Imaging

Abstract

Single molecule localization microscopy based on photoactivation is a powerful tool for investigating the ultrastructure of cells. We developed a general strategy for photoactivatable fluorophores, using 2,3dihydro-1,4-oxathiine group (SO) as a tag to attach to various skeletal structures, including coumarin, BODI-PY, rhodamine, and cyanine. The conjugation of SO resulted in a significant loss of fluorescence due to photoinduced electron transfer (PeT). Under the irradiation of excitation light, singlet oxygen generated by the fluorophores converted the SO moiety into its ester derivative, terminated the PeT process, and restored the fluorescence. Single molecule localization imaging was achieved using a dual functional illuminating beam in the visible, acting as both the activating and the exciting source. We successfully applied these photoactivatable probes for time-lapse super-resolution tracking in living cells and super-resolution imaging of microtubule structures in neurons.

Near-Infrared Spontaneously Blinking Fluorophores for Live Cell Super-Resolution Imaging with Minimized Phototoxicity

“…Given the potential of the fluorophore’s chromophore to generate reactive oxygen species (ROS) , and induce photothermal effects that might impact cell viability, we conducted a comprehensive evaluation of phototoxicity. This assessment encompassed both untreated cells and cells incubated with SMI-NIR1 after irradiation with 721 nm light at an intensity of 3.26 kW/cm 2 (as depicted in Figure a).…”

“…7 For light-activated SMLM, ultraviolet light, which is extremely phototoxic, has been traditionally used as activation light. 10 By optimizing of the chemical structure of fluorophores, the photoactivation wavelength can be shifted to the visible region, significantly improving the biocompatibility of these fluorescent dyes. 11 In addition, pH-responsive rhodamine dyes that spontaneously blink between fluorescent and nonfluorescent forms have been developed for SMLM.…”

“…The strategy has been proposed to reduce phototoxicity by shifting the irradiation wavelength to the visible region through probe chemistry . For light-activated SMLM, ultraviolet light, which is extremely phototoxic, has been traditionally used as activation light . By optimizing of the chemical structure of fluorophores, the photoactivation wavelength can be shifted to the visible region, significantly improving the biocompatibility of these fluorescent dyes .…”