This study employed nonlinear microscopy on fresh, unstained and unfixed collecting lymphatic vessels to determine the wall structure and its relationships to the mechanical properties of the tissue. Fresh bovine mesenteric collecting lymphatic vessels were mounted in a vessel bath and imaged under different luminal pressures (0-30 cmH(2)O pressure head), and longitudinal tensions. The entire wall thickness was imaged, using two-photon fluorescence to visualize elastin, second harmonic generation to image the collagen, and coherent anti-Stokes Raman scattering to image the cell membrane. The adventitial fat cells were coupled to the wall within the elastin-rich network of fibres. The medial smooth muscle cells were too densely packed to resolve the boundaries of individual cells in en face images, but in tissue sections their appearance was consistent with electron microscopic data. Two distinct populations of collagen fibre were revealed. Large fibre (15-25 mu m diameter) bundles were present in the inner media and small fibres (2-5 mu m diameter) were distributed throughout the wall. The responses to longitudinal tension and luminal pressure indicated that the larger fibres resist the longitudinal strain and the smaller oppose pressure forces. Individual elastin fibres were of uniform thickness (1-3 mu m) and interwove amongst themselves and between the collagen fibres. The network was probably too sparse directly to support mechanical loads and we speculate that its main function is to maintain the organization of collagen bundles during recovery from contraction.
- coherent anti-Stokes Raman scattering
- second harmonic generation
- RAMAN SCATTERING MICROSCOPY
- CARS MICROSCOPY