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.
Highlights
Immunohistochemical approaches to study viral pathogenesis can be highly informative but they require harsh chemical fixation as well as thin sectioning of tissue because of the poor depth penetration of traditional light microscopy
Production of vUs7-8mCherry For our pathogenesis studies of Herpes simplex virus 1 (HSV-1), we produced and characterized a recombinant strain of HSV-1 that behaves like the wild-type parental strain KOS, and that stably expresses the second generation red fluorescent protein (RFP) mCherry [39] (Figure 1)
An mCherry expression cassette under control of a mammalian promoter was inserted into the intergenic region between the open reading frames (ORFs) of the HSV-1 Us7 and Us8 genes
Summary
Immunohistochemical approaches to study viral pathogenesis can be highly informative but they require harsh chemical fixation as well as thin sectioning of tissue because of the poor depth penetration of traditional light microscopy. Both of these treatments have been demonstrated to affect tissue morphology [1], and the three-dimensional structure of the tissue is necessarily lost. Two-photon microscopy allows imaging deep (1 mm) in tissue such that intrinsically fluorescent proteins expressed within tissue can be imaged at high resolution in a three dimensional tissular context. Optimization of the experimental conditions for each imaging modality, as well as their compatibility, is crucial if one is to combine their respective strengths to achieve multimodal high spatial resolution imaging deep in tissue
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