Journal of BIOPHOTONICSWe report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 mm, while in the transverse direction it is $1-15 mm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nanocylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced.A cross section image representing a laser beam focussed in connective tissue. Collagen fibrils appear as cylinders with a random positive or negative polarity (shown with red or green). The laser intensity profile shows the size of the focal spot.

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.

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Abstract:We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

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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.

2 AbstractPurpose: To evaluate the potential of a bioactive coating based on chondroitin sulfate (CS) and tethered epidermal growth factor (EGF) for the improvement of healing around stent-grafts (SG). Methods:The impact of the bioactive coating on cell survival was tested in vitro on human vascular cells using polyethylene terephtalate films (PET) as a substrate. After being transferred onto a more "realistic" material (expanded poly(tetrafluoroethylene), ePTFE), the durability and mechanical behavior of the coating in addition to cell survival were studied. Preliminary in vivo testing was performed in a canine iliac aneurysm model reproducing type I endoleaks (3 animals with 1 control and 1 bioactive SG for each). Results:The CS and EGF coatings significantly increased survival of human smooth muscle cells and fibroblasts, compared with bare PET or ePTFE (P<.05). The coating also displayed good durability over 30 days according to ELISA and cell-survival tests.The coating did not affect the mechanical properties of ePTFE and was successfully transferred onto commercial SG for in vivo testing. According to CT-scan and macroscopic examinations, no difference was observed in endoleak persistence at three months, but the bioactive coating deposited on the abluminal surface of the SG (exposed to the vessel wall) increased the percentage of healed tissue in the aneurysm. Moreover, no adverse effect such as neointima formation or thrombosis was observed. Conclusion:The bioactive coating promoted in vitro cell survival, displayed good durability and was successfully transferred onto a commercial SG. Preliminary in vivo results suggest improved healing around bioactive SG.3