High throughput isotropic resolution fluorescence and second Harmonic imaging to characterize tendon injury - The objective of this application is to develop advanced multiview (MV) deconvolution tomography of cleared tissue using high resolution two photon microscopy (TPM) and utilize it to shed new mechanistic insights about the myofibroblast microenvironment in fibrovascular peritendinous adhesions. Advances to our understanding of the cellular and molecular events involved in tendon adhesion using transgenic mouse models, which could lead to disease modifying therapies, are often hindered by reliance on 2D histology sections and single-cell analysis techniques, neither of which can put cellular interactions or molecular processes into their 3D spatial context. Alternatively, microscopy techniques such as TPM and light sheet microscopy (LSM) can provide spatial context, but struggle to simultaneously provide the throughput, contrast and resolution required to map the cellular processes involved in tissue healing over physiologically relevant spatial and resolution scales and so are not widely used in tendon biology. To overcome these limitations, we have developed protocols for immunolabeling and tissue clearing of tendon injury models and demonstrated molecular and second harmonic generation (SHG) imaging of tendon in situ. Our proposed advances in detection electronics and imaging enable two photon microscopy two orders of magnitude faster than conventional microscopes and multicolor imaging at rates comparable to or exceeding LSM. Finally, we propose a novel approach to MV deconvolution, which simultaneously acquires multiple angular views to decrease focal volumes by an order of magnitude, enabling isotropic resolution tomographic imaging of tissues. We combine these technical advances into a high throughput platform for 3D spatial immunophenotyping and proteomics and then apply it to understanding the complex mosaic of vascular and inflammatory cells, extracellular matrix (ECM), and signaling (TGF- β1/mTOR) in the myofibroblast microenvironment, to shed new mechanistic insights that could lead to discovery of cellular and molecular targets for biologic therapies. The aims for this proposal are: Aim 1 will develop a new approach to tomography using MVD based on simultaneously imaging two angular views that will accelerate imaging and reduce the extreme computational complexity required to fuse two sequentially acquired volumes together. Eight channel acquisition will enable dense spectral multiplexing of probes and a deconvolution, enabling efficient quantification of spatial relationships between reporters at sub-wavelength scale. Aim 2 will apply MV-TPM to tomographically profile the myofibroblast microenvironment to build spatial cellular interaction networks of innate and adaptive immune cells within the vascular inflammatory microenvironment of myofibroblasts. Aim 3 will utilize MV-TPM imaging to map TGF-β1/mTOR signaling in the myofibroblast microenvironment and subsequently inhibit it to validate its role in adhesions. When completed, the proposed work will develop a high throughput 3D spatial immunophenotyping and proteomics imaging platform to shed novel mechanistic insights and discover new biologic therapies.
