Cardiac optical mapping, providing mechanistic insight into electromechanical function, is a powerful technology for studying cardiovascular activity in health and disease. The two key physiological coupled variables during the working cycle of the heart are: i) the membrane potential (i.e. electrical activity) that triggers ii) myocardial contraction (i.e. mechanical activity), in the so-called excitation-contraction coupling (ECC). At present, the most effective optical probes for membrane potential are voltage-sensitive dyes, but in most cases their response is weak and obscured by background fluorescence, keeping in mind that those cells display hyperpolarization at the resting state. Mechanosensitive fluorophores appeared recently and, to the best of our knowledge, they have not been used for applications in sensing of membrane strain in cardiac cells.
We will exploit the complementary expertise of the staff members in the fields of: i) design and optical properties of fluorophores; ii) organic and supramolecular synthesis; iii) physiology of cardiomyocytes; iv) multiphoton microscopy; to prepare innovative optical probes for sensing membrane potential and membrane tension with high spatio-temporal resolution.
Two novel mechanisms for biosensing will be exploited. The symmetry lowering of symmetric chromophores experiencing an electric field will affect their fluorescence properties, allowing to probe the membrane potential. Fluorescence resonance energy transfer (FRET) between two chromophores grafted on a flexible scaffold, whose distance can be affected by mechanical forces, will be exploited to probe the membrane tension.
We will image the cells and tissues via two-photon (2P) microscopy, coupled with spectral detection, to sense the membrane potential and tension in the 3D space, with sub-micrometer resolution, and in time, with sub-millisecond resolution. Multiphoton imaging techniques are consolidated, but sensing is typically performed without 3D spatial resolution. Our ambitious goal is to reach a 4D-resolved sensing, able to describe in space and in time the working mechanism of cells.
Towards this aim, we will design voltage-sensitive and mechano-sensitive fluorophores and multifluorophores optimized for 2P-absorption, as to gain sensitivity in the detection of emission spectra and their changes induced by variations of potential or strength, thus improving the signal-to-noise ratio.
These dyes, being specifically designed for nonlinear optics, shall also have strong second harmonic generation (SHG) capability, opening the possibility to image the samples and sense the aimed variables in an alternative way, which could have even higher sensitivity thanks to SHG only stemming from asymmetric environments (such as membranes).