Spectroscopic Fluorescence Microscopy Approaches for Real-Time Visualization of Viral Infection Dynamics in Living Cells

The virus–cell interaction processes are extremely dynamic. Since viruses lack the necessary machinery for energy production and replication, they are entirely dependent on host cells to generate viral progeny. During this interaction, several natural cellular mechanisms are hijacked by the virus, which, upon releasing its nucleic acid, alters the cell's function to exploit the intracellular environment for its own replication. In this context, new molecular patterns are produced, novel structures are formed, and significant changes occur in the metabolism and organization of the infected host cell. Studying these processes in living cells remains a major challenge in virology and cell biology. Many traditional techniques require either purification of components or the fixation of cells for visualization. Identifying specific components in living cells, such as viral RNAs, studying viral factory formation involving membranous structures, and monitoring dynamic changes in the cellular environment during viral infection and replication remain significant challenges for researchers. Through the use of optical microscopy and environment-sensitive fluorescent probes, such as solvatochromic dyes, it is possible to monitor these processes in living cells with minimal perturbation. Spectroscopic methods adapted for microscopy allow the acquisition of hyperspectral images and fluorescence lifetime imaging (FLIM). With the appropriate selection of fluorescent probes, information can be obtained regarding the dynamics and physicochemical properties of lipid membranes (Laurdan), the distribution of nucleic acids (Pyronin Y), and the metabolic state of infected cells (NAD(P)H, FAD). Data analysis using novel approaches, such as the Phasor method, facilitates the interpretation of these complex datasets and enables the direct observation of viral infection processes. Using fluorescence spectroscopy approaches combined with microscopy, we have investigated the distribution of double-stranded RNA during arbovirus infection, the remodeling of cellular membranes, the formation and stability of viral envelopes, and the energetic metabolism of cells infected by different viruses. Our results highlight the power of fluorescence microscopy and spectroscopy to advance the real-time understanding of viral infections in living systems.