Quaternary Piperazine-Substituted Rhodamines with Enhanced Brightness for Super-Resolution Imaging

Abstract

Insufficient brightness of fluorophores poses a major bottleneck for the advancement of super-resolution microscopes. Despite being widely used, many rhodamine dyes exhibit sub-optimal brightness due to the formation of twisted intra­molecular charge transfer (TICT) upon photoexcitation. Herein, we have developed a new class of quaternary piperazine-substituted rhodamines with outstanding quantum yields (Φ = 0.93) and superior brightness (ε × Φ = 8.1 × 104 L·mol–1·cm–1), by utilizing the electronic inductive effect to prevent TICT. We have also successfully deployed these rhodamines in the super-resolution imaging of the microtubules of fixed cells and of the cell membrane and lysosomes of live cells. Finally, we demonstrated that this strategy was generalizable to other families of fluorophores, resulting in substantially increased quantum yields.

A General Strategy to Enhance Donor‐Acceptor Molecules Using Solvent‐Excluding Substituents

“…Instead, calculation of the dipole moment of the excited and ground states (μ E and μ G , respectively) indicates a lesser overall difference between these values with β‐carbonyl functionalization, supporting a model wherein intramolecular charge transfer in the excited state is inhibited (see Figure S6c). This result suggests that the increase in requisite energy for rotation is caused by the inductive electron‐withdrawing nature of the carbonyl group, which would give rise to a destabilization of the amino radical cation of the TICT state . This is further supported by the lesser overall difference in the driving energy between LE and TICT states of 3 (3.50 kcal mol −1 ) compared to that of 1 (6.72 kcal mol −1 ; Figure ).…”

Descriptor ΔGC‐O Enables the Quantitative Design of Spontaneously Blinking Rhodamines for Live‐Cell Super‐Resolution Imaging

“…The formation of the TICT state is a major non‐radiative decay mechanism that deteriorates the quantum yields of rhodamines [17] . Suppressing TICT formation enhances the quantum yields and improves the overall brightness of fluorophores [4c, 18] . This enhancement can be achieved either by reducing the electron‐donating strength of the amino group (R; Figure 1 a) and/or adjusting steric hindrance. Incorporation of handles for easy functionalization .…”

Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging

“…In superresolution imaging where uorophore brightness is of pivotal importance and in primary-cell analysis where genetic manipulations are cumbersome (if not impossible), organic dyes are indispensable reagents. In recent years, innovation in bioimaging dyes has mainly focused on brightness, 27,40,41 uorogenicity, 42,43 photostability, 7,44,45 and photo-switching. 5 Despite similarities in their mechanism, the distinctions between phototoxicity and photobleaching are important in uorophore design.…”