Ismael López-Duarte

Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors

In order to fully understand the dynamics of processes within biological lipid membranes, it is necessary to possess an intimate knowledge of the physical state and ordering of lipids within the membrane. Here we report the use of three molecular rotors based on meso-substituted boron-dipyrrin (BODIPY) in combination with fluorescence lifetime spectroscopy to investigate the viscosity and phase behaviour of model lipid bilayers. In phase-separated giant unilamellar vesicles, we visualise both liquid-ordered (Lo) and liquid-disordered (Ld) phases using fluorescence lifetime imaging microscopy (FLIM), determining their associated viscosity values, and investigate the effect of composition on the viscosity of these phases. Additionally, we use molecular dynamics simulations to investigate the orientation of the BODIPY probes within the bilayer, as well as using molecular dynamics simulations and fluorescence correlation spectroscopy (FCS) to compare diffusion coefficients with those predicted from the fluorescence lifetimes of the probes.

Imaging tumor microscopic viscosity in vivo using molecular rotors

The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior. While several approaches have emerged that have allowed the measurement of viscosity and diffusion on a single cell level in vitro, the in vivo viscosity monitoring has not yet been realized. Here we report the use of fluorescent molecular rotors in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to image microscopic viscosity in vivo, both on a single cell level and in connecting tissues of subcutaneous tumors in mice. We find that viscosities recorded from single tumor cells in vivo correlate well with the in vitro values from the same cancer cell line. Importantly, our new method allows both imaging and dynamic monitoring of viscosity changes in real time in live animals and thus it is particularly suitable for diagnostics and monitoring of the progress of treatments that might be accompanied by changes in microscopic viscosity.

“Push-no-pull” porphyrins for second harmonic generation imaging

The established approach to design a molecule with strong second-order nonlinear optical (NLO) activity is to connect an electron-donor to an electron-acceptor via a π-conjugated bridge, to generate push–pull system. Surprisingly, we have found that dyes with large first hyperpolarizabilities, and which exhibit strong second harmonic generation (SHG), can be created just by attaching an electron-donor to a porphyrin. The free-base porphyrin core is sufficiently electron-deficient that the hyperpolarizability does not increase on addition of a pyridinium electron-acceptor.

Imaging plasma membrane phase behaviour in live cells using a thiophene-based molecular rotor

Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.