Multi-Photon Fluorescence Imaging through Biological Tissue and Image Reconstruction

In this paper, image formation under single-photon (1-p), two-photon (2-p) and three-photon (3-p) fluorescence imaging through turbid media which consist of different sized scatterers has been investigated in detail. It has been demonstrated that the size of scattering particles plays an important role in determining whether to use 1-p, 2-p, or 3-p excitation. For small scatterers, where Rayleigh scattering is dominant, multi-photon excitation provides significantly better resolution. Such improvement reduces dramatically for large scatterers, where Mie scattering becomes dominant. Another disadvantage of using multi-photon fluorescence excitation in highly scattered media is that penetration depth is limited by fast dropping of signal strength in deep tissue imaging. In this paper, we introduce a deconvolution method with a novel concept of the effective point spread function, which is effective in restoring the loss of imaging resolution caused by multiple scattering in a tissue medium.