Ricerc@Sapienza

The in-vivo study of physiological and pathological events occurring in inner organs is limited by their intrinsic accessibility. Over time, scientists have engineered ways around this limitation by indirect manipulation. Common approaches combine fluorescent imaging and recording techniques to isolated organs, tissues and cells, mimicking the best "body-like" environments. These strategies capitalize also by innovative genetic and chemogenetic techniques allowing the selective targeting of specific gene products or allowing the on/off-switching of their expression at the embryonal or postnatal age. However, the direct in vivo imaging from inner organs in their physiological context by one-photon absorption based fluorescence microscopy, is not possible. This is because the heterogeneous composition of biological tissues is responsible of a strong light scattering, and the quality of contrast depends linearly on the incident light intensity. The so called non-linear optical microscopy, and in particular the two photon microscopy (2PM), provides the opportunity to overcame this limit, making high-resolution deep imaging possible through long-wavelength laser sources that reduce light scattering. The combination of 2PM and in vivo fluorescence labelling coupled to electrophysiology gives the possibility to study intact tissues in living animals, obtaining imaging at depths of more than 1 mm from organs such as the heart, kidney, lymphatic organs, muscle, and brain, performing functional studies even in awake animals. This represented a revolutionary technical progress for the study of not easily accessible systems such as the CNS, where the 2PM permitted seminal discoveries in the last two decades, from dendritic spine plasticity during learning (Trachtenberg et al. Nature 2002) to homeostatic activity of microglial cells (Davalos et al Nat Neurosci 2005).