Abstract
Cytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. We report distinct patterns of virus infections in live cells using digital holo-tomographic microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the specimen on a transparent surface with minimal perturbation. DHTM measures the refractive index (RI) and computes the refractive index gradient (RIG), unveiling optical heterogeneity in cells. We find that vaccinia virus (VACV), herpes simplex virus (HSV), and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV infection, but not HSV and RV infections, induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics and induced apoptotic features akin to those caused by the chemical compound staurosporine. In sum, we introduce DHTM for quantitative label-free microscopy in infection research and uncover virus type-specific changes and CPE in living cells with minimal interference.IMPORTANCE This study introduces label-free digital holo-tomographic microscopy (DHTM) and refractive index gradient (RIG) measurements of live, virus-infected cells. We use DHTM to describe virus type-specific cytopathic effects, including cyclic volume changes of vaccinia virus infections, and cytoplasmic condensations in herpesvirus and rhinovirus infections, distinct from apoptotic cells. This work shows for the first time that DHTM is suitable to observe virus-infected cells and distinguishes virus type-specific signatures under noninvasive conditions. It provides a basis for future studies, where correlative fluorescence microscopy of cell and virus structures annotate distinct RIG values derived from DHTM.
Highlights
Cytopathic effects (CPEs) are a hallmark of infections
We describe a new approach using 3D digital holo-tomographic microscopy (DHTM) to study the CPEs induced by three different viruses: vaccina virus (VACV), a large DNA virus replicating in the cytoplasm, herpes simplex virus type 1 (HSV-1), a large DNA virus replicating in the nucleus, and rhinovirus (RV), a small RNA virus replicating on cytoplasmic membranes
The uninfected cells and the vaccinia virus (VACV)-GFP-infected cells treated with the deoxynucleoside analogue cytarabine (AraC), an inhibitor of VACV late gene expression [16, 17], showed less prominent refractive index (RI) changes, AraC-treated cells inoculated with VACV-GFP exhibited a strong increase in RI in the cell nucleus
Summary
Cytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. IMPORTANCE This study introduces label-free digital holo-tomographic microscopy (DHTM) and refractive index gradient (RIG) measurements of live, virus-infected cells. We use DHTM to describe virus type-specific cytopathic effects, including cyclic volume changes of vaccinia virus infections, and cytoplasmic condensations in herpesvirus and rhinovirus infections, distinct from apoptotic cells. This work shows for the first time that DHTM is suitable to observe virus-infected cells and distinguishes virus type-specific signatures under noninvasive conditions It provides a basis for future studies, where correlative fluorescence microscopy of cell and virus structures annotate distinct RIG values derived from DHTM. Label-free digital holo-tomographic microscopy across cells in 3 dimensions reveals virus-specific features, including periodic cell volume changes, and cytoplasmic membrane dynamics for vaccinia virus, herpesvirus, and rhinovirus @ayakimovich. Despite the predictive nature of CPE for clinical and biological infections, time-resolved 3D analyses of virus-induced CPE are missing
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