Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal‐based cholesterol probe (chol‐N3) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol‐N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live‐cell super‐resolution microscopy, chol‐N3 demonstrates the existence of cholesterol‐rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.

The Raman phenomenon is based on the spontaneous inelastic scattering of light, which depends on the molecular characteristics of the dispersant. Therefore, Raman spectroscopy and imaging allow us to obtain direct information, in a label-free manner, from the chemical composition of the sample. Since it is well established that the development of many brain diseases is associated with biochemical alterations of the affected tissue, Raman spectroscopy and imaging have emerged as promising tools for the diagnosis of ailments. A combination of Raman spectroscopy and/or imaging with tagged molecules could also help in drug delivery and tracing for treatment of brain diseases. In this review, we first describe the basics of the Raman phenomenon and spectroscopy. Then, we delve into the Raman spectroscopy and imaging modes and the Raman-compatible tags. Finally, we center on the application of Raman in the study, diagnosis, and treatment of brain diseases, by focusing on traumatic brain injury and ischemia, neurodegenerative disorders, and brain cancer.

Interorganelle membrane contact sites (MCS) are areas of close vicinity between the membranes of two organelles that are maintained by protein tethers. Recently, a significant research effort has been made to study MCS, as they are implicated in a wide range of biological functions, such as organelle biogenesis and division, apoptosis, autophagy, and ion and phospholipid homeostasis. Their composition, characteristics, and dynamics can be studied by different techniques, but in recent years super-resolution fluorescence microscopy (SRFM) has emerged as a powerful tool for studying MCS. In this review, we first explore the main characteristics and biological functions of MCS and summarize the different approaches for studying them. Then, we center on SRFM techniques that have been used to study MCS. For each of the approaches, we summarize their working principle, discuss their advantages and limitations, and explore the main discoveries they have uncovered in the field of MCS.

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