White‐Fluorescent Dual‐Emission Mechanosensitive Membrane Probes that Function by Bending Rather than Twisting

Abstract

Bent N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine amphiphiles are introduced as mechanosensitive membrane probes that operate by an unprecedented mechanism, namely, unbending in the excited state as opposed to the previously reported untwisting in the ground and twisting in the excited state. Their dual emission from bent or “closed” and planarized or “open” excited states is shown to discriminate between micelles in water and monomers in solid‐ordered (So), liquid‐disordered (Ld) and bulk membranes. The dual‐emission spectra cover enough of the visible range to produce vesicles that emit white light with ratiometrically encoded information. Strategies to improve the bent mechanophores with expanded π systems and auxochromes are reported, and compatibility with imaging of membrane domains in giant unilamellar vesicles by two‐photon excitation fluorescence (TPEF) microscopy is demonstrated.

Fluorescent Flipper Probes: Comprehensive Twist Coverage

“…In clear contrast, increasing viscosity in solution caused only marginal shifts of the excitation maximum of CF 3 flipper 2′ (Figure c). This control supported that the redshifted excitation of flipper probes in membranes of increasing order originates from mechanical planarization in equilibrium in the ground state, and not from off‐equilibrium kinetics . Contrary to original flipper 1 , fluorescence intensities of CF 3 isostere 2 increased with increasing viscosity (Figures S5, S6).…”

Mechanosensitive Fluorescent Probes to Image Membrane Tension in Mitochondria, Endoplasmic Reticulum, and Lysosomes

“…Consistent with increasing planarization in the ground state, flippers 2-4 gave the expected red-shifted excitation maxima in solid-ordered (So) compared to liquid-disordered (Ld) membranes of large unilamellar vesicles (LUVs, Figures 2A, S1-S9, Table 1). 7,9,[19][20][21] Also as expected were the higher average lifetimes t of 2-4 in liquid-ordered (Lo) compared to Ld membranes in FLIM images of giant unilamellar vesicles (GUVs, Figures 2B, S11, Table 1). 5,19 Compared to the meticulously optimized original 1 19 in ordered membranes (lSo 490 nm, tLo 6.4 ns), the new probes 2-4 showed overall smaller red shift of the excitation wavelength (lSo 475 nm) and the lifetime (tLo 5.5-5.8 ns), presumably due to less perfect positioning along the lipid tails in the membrane (Table 1).…”

“…5 Such properties are unique 9 among the membrane probes, that usually report off-equilibrium in the excited state on viscosity or polarity. [9][10][11][12][13][14][15][16] In cellular membranes, the mechanophore 1 reports the increasing membrane order upon application of membrane tension s by increasing fluorescence lifetime t. This change was attributed to the formation of more ordered microdomains under membrane tension, due to the increased line tension between the stretchable and unstretchable lipid phases ( Figure 1B, dark gray). 5,17 Roughly linear positive t-s correlations found with all tested cells demonstrated the applicability of 1 to measure membrane tension change by fluorescence lifetime imaging microscopy (FLIM).…”

“…All values were inbetween the extremes measured in pure single-component model Ld and Lo membranes in GUVs. The variations in lifetimes originate mainly from differences in membrane organization (including contributions from lipid composition, microdomains, maybe also proteins, viscosity, etc), 5,9,10,23 and from the different mechanosensitivity of probes 1-4 ( Figure 2A, Table 1). Thus, lifetimes in organelles at rest cannot be directly correlated to possible differences in membrane tension.…”

Dithienothiophenes at Work: Access to Mechanosensitive Fluorescent Probes, Chalcogen-Bonding Catalysis, and Beyond

“…In bent rather than twisted systems, it has been shown to afford ratiometrically-encoded mechanosensitive white emission. [91][92][93] With mesogenic substituents R in 125, red, orange-fluorescent gels are obtained.…”