Intrinsic optical biomarkers associated with the invasive potential of tumor cells in engineered tissue models

This report assesses the ability of intrinsic two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to characterize features associated with the motility and invasive potential of epithelial tumor cells engineered in tissues. Distinct patterns of organization are found both within the cells and the matrix that depend on the adhesive properties of the cells as well as factors attributed to adjacent fibroblasts. TPEF images are analyzed using automated algorithms that reveal unique features in subcellular organization and cell spacing that correlate with the invasive potential. We expect that such features have significant diagnostic potential for basic in vitro studies that aim to improve our understanding of cancer development or response to treatments, and, ultimately can be applied in prognostic evaluation

The elastin network: its relationship with collagen and cells in articular cartilage as visualized by multiphoton microscopy

A combination of two-photon fluorescence (TPF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been used to investigate the elastin fibre network in healthy equine articular cartilage from the metacarpophalangeal joint. The elastin fibres were identified using their intrinsic two-photon fluorescence and immuno-staining was used to confirm the identity of these fibres. SHG was used to reveal the collagen matrix and the collagen fibre orientations were determined from their SHG polarization sensitivity, while CARS was used to clearly delineate the cell boundaries. Extensive elastin fibre networks were found in all the joint regions investigated. The elastin was found predominantly in the superficial zone (upper 50 μm) and was aligned parallel to the articular surface. Elastin was also detected in the pericellular matrix surrounding the superficial chondrocytes; however, individual fibres could not be resolved in this region. Variations in the density and organization of the fibres were observed in different regions on the joint surface

Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications