Fluorescence Ratiometry and Fluorescence Lifetime Imaging: Using a Single Molecular Sensor for Dual Mode Imaging of Cellular Viscosity

Abstract

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (λ(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, λ(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and (1)H NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.

A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress

“…In this work, we utilized BODIPY‐C 10 17, 18 (Figure 1), a fluorophore that belongs to a group of dyes termed ‘molecular rotors’ that have viscosity‐dependent fluorescence quantum yields, lifetimes,19, 20 and depolarization 21, 22. When combined with fluorescence lifetime imaging microscopy (FLIM), molecular rotors can be used to obtain spatially resolved viscosity maps of microscopic objects,17, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 as well as to observe dynamic change in viscosity during relevant processes of interest 37, 39, 41, 42. Thus, we aimed to use BODIPY‐C 10 , which is known to completely embed into the fluid‐phase lipid bilayers40 to directly examine how photooxidation during PDT affects viscoelastic properties of model lipid membranes, with spatial‐ and time‐resolution.…”

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

“…It is now well established that quantitative imaging of viscosity requires either ratiometric detection or fluorescence lifetime measurements, in order to rule out the effect of the concentration of the probe on the observed signal. [16][17][18][19] Consequently, both types of molecular rotors, using either ratiometric or lifetime detection, have been reported and used for imaging in a variety of systems, from live cells to lipid membranes to atmospheric aerosols. 2,[16][17][18]20 Here we report a red emitting molecular rotor 1, Figure 1a, previously synthesized by Balaz et.…”

Tuning the Sensitivity of Fluorescent Porphyrin Dimers to Viscosity and Temperature

“…Such a dual sensing approach is beneficial, since it allows us to independently verify the viscosity read‐out using two independent measurements 18, 30. Here, we tested whether the dimers 1 , 3 and 4 also have viscosity‐sensitive fluorescence decays.…”