CONCURRENT SECOND HARMONIC GENERATION (SHG) IMAGING OF COLLAGEN ORIENTATION AND TWO-PHOTON FLUORESCENCE CELL IMAGING MICROSCOPY IN TENDONS

Summary Objective assessment of tendon histomorphology, particularly in the context of tissue repair, requires comprehensive analyses of both cellular distribution and matrix architecture. Fourier Transform analyses of histological images collected with second harmonic generation (SHG-FT) technique provide objective, quantitative assessment of collagen fiber organization with high specificity. Concurrent nuclear staining allows simultaneous analyses of cell morphology and distribution. Introduction Tendon injuries can be career-limiting in human and equine athletes, since the architectural organization of the tissues are lost in the course of fibrotic repair. Objective assessment of tendon repair is problematical, particularly in research addressing potential therapies. Fourier Transform analyses of histological images collected with second harmonic generation (SHG-FT) technique can provide objective, quantitative assessments of collagen fiber organization with high specificity. This study describes the use of SHG-FT with fluorescently-labelled tendon-derived cells (TDC) in an in-vivo model of equine tendinitis to assess the temporal and spatial effects of cell delivery on collagen fiber organization. Materials and methods Collagenase-induced tendinitis was created in the mid-metatarsal region of one hindlimb superficial digital flexor tendons (SDFT) in two horses. SDFTs from two clinically normal adult horses and were also used as controls. Autogenous TDCs were isolated from the lateral digital extensor tendon of the contralateral hind limb. Four weeks post-collagenase injection, 10×10 6 DiI-labeled TDCs were injected into the tendon lesions. Tendon samples were obtained for histologic evaluation following euthanasia, 2-weeks after cell injections. Tendon samples were cryo-sectioned to 25–30μ exposed to nuclear counter stains (DAPI and PI) and imaged immediately through a confocal microscope (Zeiss LSM 710) with a 2-photon laser source, to obtain backward SHG (bSHG) and forward SHG (fSHG) images. In addition, images with DiI and DAPI fluorescence were acquired using 500–550 nm (green) or 565–615 nm (orange) emission filters, respectively. Fourier analysis of the SHG images was carried out using imageJ software. Results DiI-labeled TDCs could be imaged successfully under two-photon fluorescence concurrently with SHG imaging. This was possible because the excitation wavelength of the two-photon laser (780nm) and detection of emissions above 565nm do not interfere with the bSHG band (380–400nm). Images collected with bSHG included signals from DAPI-stained nuclei. In contrast, emissions from PI-labeled nuclei were acquired independently of SHG signals. The contrast generated by individual collagen fibers was higher in images collected with fSHG than bSHG. SHG-FT of fSHG images provided accurate assessment of collagen fiber orientation in repair tissue and normal tendon. Discussion/Conclusions Objective assessment of collagen orientation, along with spatial distribution of cells within healing tendon serves as useful indices of healing. Injected DiI-labeled TDCs could be imaged successfully under two-photon fluorescence concurrently with SHG imaging. However, DiI fluorescence is susceptible to photo-bleaching during SHG acquisition. Use of an alternative nuclear counter stains, such as PI, that do not emit along with SHG signal should be considered to optimise data acquisition and support simultaneous analyses of collagen structure, cellular morphology and cell distribution. SHG-FT histologic analysis along with biochemical and biomechanical indices collectively provide comprehensive assessment of therapies for tendon repair.