In tissue engineering, a scaold provides the necessary framework for cells to grow into the desired three-dimensional (3D) tissues or organs (Sachlos et al., 2003). e ability to capture the 3D dynamic of the growing process calls for a minimally invasive imaging modality with 3D imaging at sucient resolution to identify the scaold and the cell components of a tissue engineering construct. e ability of a multiphoton microscope to simultaneously register multiple spectral uorescence signals and the second-harmonic generation (SHG) signal provides a rich amount of information for both scaold and cell component characterization. e use of a long excitation wavelength and a no-pinhole detection scheme decreases the sample scattering eect both in the beam excitation path and in the signal detection path. e minimally invasive nature of multiphoton microscopy together with its intrinsic optical sectional capability makes it particularly suitable for capturing long-term 3D dynamics in tissue engineering.