Diffusion measured by fluorescence recovery after photobleaching based on multiphoton excitation laser scanning microscopy.

摘要: Fluorescence recovery after photobleaching (FRAP) is a widely used method to measure diffusion. The technique normally based on one-photon excitation, which limits diffusion two dimensions due extended in the axial direction. Multiphoton other hand, creates well-defined focal volume. In present work, FRAP scanning laser beam and two-photon excitation of macromolecules solution gels, as well extracellular matrix multicellular spheroids tumor tissue dorsal chambers. bleaching profile determined experimentally immobilized for small areas (approximately twice lateral radius beam) Gaussian distribution found. addition, theoretically convolution point spread function circular area. coefficient by fitting mathematical model experimental curve. decreases with increasing complexity sample amount collagen gels. potential using microscopes noninvasive measurements demonstrated.