We have successfully engineered a recombinant HSV-1 strain that expresses mCherry all the while retaining a wild-type phenotype in vivo. Furthermore, we found that the virus is stable both in cell culture and following passage through mice. Thus, this virus constitutes one of a limited number of recombinant HSV-1 strains that encode an intrinsically fluorescent protein, and that are suitable for in vivo studies looking at pathogenesis. mCherry was an excellent choice for visualization of infected cells in situ due to the low natural fluorescence of biological samples corresponding to its visual spectrum. The mCherry signal was sufficiently above the autofluorescence background with limited photobleaching to detect both the cell bodies and the axons of many infected neurons in the tissue. Further improvement to our strategy would include having an even stronger signal to noise ratio for the multiphoton fluorescent imaging. One approach would be the use of the RFP tdTomato, for which the gene is a duplicated Molsidomine mTomato ORF in tandem and in frame. This intramolecular dimer is three times more brilliant than mCherry, and has been detected as deep as 1 cm within tissues, making it ideal for the study of viral dissemination within an experimental animal. Finally, using the sectioning power of our microscopy platform, we recreated a three-dimensional projection of an ROI deep within the unfixed TG where infected neurons were located. In this projection, infected neurons and axons could be observed in relation to their native environment. From there, we were able to select ROIs to analyze in more Pantethine detail combining fluorescence and CARS microscopy techniques. Our results demonstrate the potential of multimodal non-linear optical microscopy for studying viral infection and pathogenesis in whole tissues by combining data acquired through different imaging modalities. Furthermore, because CARS imaging is particularly well suited to studying demyelinating diseases, our platform would be ideal for investigating hypotheses of virus-induced demyelination among other virus-induced neuropathologies.