"Development of Heterodyne Brillouin Microscopy: Principles, Limitations, and Perspectives"

Mechanical properties play a fundamental role in the regulation of biological processes, and their alteration is increasingly recognized as a hallmark of disease, including neu- rodegenerative disorders. Despite their importance, the quantitative characterization of cellular and subcellular mechanics in living systems remains experimentally challenging. In this context, Brillouin microscopy has emerged as a non-contact and label-free optical technique capable of probing high-frequency viscoelastic properties with submicron spatial resolution. This thesis investigates Brillouin microscopy as a tool for mechanobiology, with a par- ticular focus on the experimental strategies used for Brillouin signal detection. After re- viewing the biological motivations and the theoretical framework of light scattering and Brillouin spectroscopy, the work presents a description of state-of-the-art Brillouin mi- croscopy implementations based on high-resolution dispersive spectrometers. A stabilized double-VIPA Brillouin microscope is described in detail and applied to the investigation of protein condensates in living cells, demonstrating the capability of the technique to address biologically and neuroscientifically relevant systems. The core contribution of this thesis is the development and systematic evaluation of a heterodyne Brillouin microscopy approach as an alternative detection paradigm. The physical principles of heterodyne detection are introduced, followed by a comprehen- sive experimental characterization of the setup, including noise analysis, signal-to-noise ratio modeling, and experimental validation. The performance of heterodyne detection is quantitatively compared with conventional VIPA-based spectrometer detection using benchmark measurements on laser sources and optical fibers. The results reveal that, while heterodyne Brillouin microscopy offers conceptual and practical advantages in terms of detection flexibility, its sensitivity in the investigated con- figurations is ultimately limited by the intrinsic manner in which signal and noise are jointly integrated within the spectral resolution bandwidth. This finding defines the fun- damental performance boundaries of the technique and provides critical insight into its domain of applicability. Overall, this work clarifies the strengths and limitations of het- erodyne Brillouin microscopy and contributes to the broader effort of developing robust optical methods for quantitative biomechanical imaging of biological systems.