Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy

Abstract

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives.

Concepts in Light Microscopy of Viruses

“…The superior penetration depth and low phototoxicity make multiphoton microscopy interesting for studying host–pathogen interactions intravitally, although logistic adjustments to match biosafety standards remain a challenge [ 188 , 189 ]. Furthermore, multiphoton microscopes allow for integrating additional imaging modalities, such as fluorescence recovery after photobleaching (FRAP) to measure diffusion kinetics of a fluorescent entity [ 190 ], fluorescence-lifetime imaging microscopy (FLIM) used for visualizing the lifetime of individual fluorophores [ 191 ], and coherent anti-Stokes Raman spectroscopy (CARS), which allows for label-free imaging based on vibrational signatures of biological samples [ 192 , 193 ].…”

Microbial pathogenesis revealed by intravital microscopy: pros, cons and cautions

“…Most intravital imaging studies visualize virus infected cells, but not virus particles themselves, by expression of a virus‐encoded fluorescent protein in infected cells . If direct fluorescent protein‐tagging of viral proteins does not affect infectivity, one virus can be used to detect input viruses (as described above) and serves as a marker for infected cells, in addition.…”

“…Agarose‐embedded, sometimes even sectioned organs were imaged in ex vivo multiphoton imaging approaches in the past . Immune cell migration, although sometimes at reduced speed compared to in vivo approaches, is retained in explanted organs perfused by oxygenated medium.…”

“…Immune cell migration, although sometimes at reduced speed compared to in vivo approaches, is retained in explanted organs perfused by oxygenated medium. Organ explants have the advantage of easily eliminating movement artefacts, such as breathing motion observed during in vivo lung imaging , and prior sectioning allows imaging deep within otherwise inaccessible parts of an organ . For some applications, organ explants may therefore represent the method of choice; however, drawbacks prevail.…”

“…Viruses of the herpesviridae family actively replicate within or withdraw into neuronal ganglia during latency. Two recent studies utilized intravital imaging of trigeminal and submandibular ganglia for imaging of HSV‐1 and Pseudorabies Virus (PRV), respectively . PRV infection causes the characteristic “mad itch” syndrome in which infected animals scratch an affected area until self‐mutilation.…”