Develpoment and Characterization of Novel Probes for Photoacoustic Microscopy

The photoacoustic effect (PA) is a physical phenomenon based on the emission of sound waves following light absorption in material samples. The photon absorption and subsequent non-radiative depletion of the chromophores rapidly rises the temperature within the sample, increasing the pressure and inducing a thermoelastic expansion, leading to the emission of a pressure wave called photoacoustic wave. The reduced scattering of acoustic waves enables high-resolution, deeply penetrating imaging in biological tissues. The difference among the absorption coefficients of tissue components or suitable transgene labels in the sample gives the base of contrast in PA imaging but the few number of probes showing high PA efficiency reduces the applications to living systems and processes at the cellular and subcellular levels. The development of reversibly switchable fluorescent proteins (rsFPs) has revolutionized the life science imaging contributing to optical nanoscopy as agents able to improve contrast-to-noise ratio and spatial resolution. The competiveness between light and heating emission in rsFPs optimized for fluorescence imaging considerably reduces the acoustic emission efficiency making them not suitable for PA microscopy. The aim of this project is the development of a novel approach in photoacoustic microscopy inspired by the toolbox of knowledge we have for fluorescence imaging. The key point lies in the probes, belonging to two different families of photochromic proteins, which will be characterized and applied in the novel photoacoustic microscope: GAF3, and two new mutants of GFPs obtained adding a fluorescence-decreasing mutation to wildQ and wildQT proteins.