Spatial frequencies from human periosteum at different depths using two-photon microscopic images

The outer layer of human bone, the periosteum, was studied using two-photon (2P) fluorescence microscopy. This layer of the periosteum is composed mostly of fibrous collagen. The inner cambium layer has less collagen and contains osteoblasts necessary for bone remodeling. The spatial frequencies from the layers of the periosteum of human bone at different depths were investigated using images acquired with two-photon excitation microscopy. This 2P spectroscopic method offers deeper depth penetration into samples, high fluorescence collection efficiency, and a reduction in photobleaching and photodamage. Using 130 femtosecond pulses with an 800 nm wavelength excitation, a 40× microscope objective, and a photomultiplier tube (PMT) detector, high contrast images of the collagen present in the periosteum at various micrometers depths from the surface were obtained. Fourier transform analysis of the 2P images was used to assess the structure of the periosteum at different depths in terms of spatial frequencies. The spatial frequency spectra from the outer and inner periosteal regions show significant spectral peak differences which can provide information on the structure of the layers of the periosteum. One may be able to use spatial frequency spectra for optical detection of abnormalities of the periosteum which can occur in disease.