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

CHIKV is a virus of the genus Alphavirus (family Togaviridae) with a positive-sense single-stranded RNA (ssRNA+) genome. During replication, significant reorganization of host cell membranes occurs, leading to the formation of viral replication complexes where ssRNA+ replication takes place, generating double-stranded RNA (dsRNA). While abundant during viral infection, dsRNA is found in low proportions in uninfected cells. Pyronin Y (PY) is a fluorescent probe that intercalates into double-stranded nucleic acids—specifically dsRNA—forming fluorescent complexes with distinct spectral signatures. Additionally, LAURDAN is a solvatochromic fluorescent probe capable of inserting into membranes. When the probe is inserted into membranes in either the gel or fluid phase, a shift in the fluorescence spectrum is observed, enabling the analysis of changes in lipid membrane organization and localization within the cell. Objective: To use PY and LAURDAN probes to track different dsRNA species within cells, identifying potential viral dsRNAs associated with replication complexes and their fates in living cells, as well as visualizing potential changes in membrane fluidity in infected cells using a spectral phasor approach. Materials and Methods: BHK-21 cells were infected with CHIKV at a multiplicity of infection (MOI) of 5. At various time points, cells were labeled, and images were acquired using a Zeiss LSM 710 ELYRA PS.1 confocal microscope. Excitation wavelengths were set at 405 nm (for LAURDAN) and 514 nm (for PY). Fluorescence emission was collected across 32 combined channels, covering the 416–728 nm wavelength range. Spectral images (512×512 pixels) were acquired for each cell. Spectral unmixing and principal component analysis were performed using SimFCS 4 software, employing the spectral phasor approach, which maps each pixel of the acquired images to x and y coordinates on a scatter plot. Results: In certain regions, infected cells exhibit PY-specific fluorescence emission at longer wavelengths compared to uninfected cells. LAURDAN analyses indicate the formation of more organized lipid membrane structures as the infection progresses. Conclusion: Preliminary results indicate that PY and hyperspectral imaging can be useful tools for studying the dynamics of double-stranded viral RNA species and membrane organization dynamics during the viral replication cycle in living cells.