Intravital imaging of gastrointestinal diseases in preclinical models using two-photon laser scanning microscopy

Two-photon laser scanning microscopy (TPLSM), relying on two-photon excitation restricted to the focal plane, has become the gold standard in biomedical research because of its ability to produce high-resolution, higher penetrating imaging of biological materials over an extended duration without an significant photobleaching. Intravital (in vivo) imaging using TPLSM for intra-abdominal organs has long been a technical challenge because of the difficulty of achieving high-quality and higher magnification imaging with acceptable cardiac and respiratory motion artifacts. A method of intravital TPLSM was developed using an organ-stabilizing system for imaging the intra-abdominal organs of green fluorescent protein transgenic mice. This method was further refined for the time-series imaging of intra-abdominal organs in the same mouse model using intravital TPLSM. These procedures allow the observation of not only a single cell or tissue microenvironment at a higher penetrating depth for a longer period of time but also to observe the same organ of the same mouse at multiple time points in preclinical models. This report presents the general principles and properties of TPLSM for biomedical research. In addition, the methods and the usefulness of time-series intravital TPLSM imaging for preclinical gastrointestinal diseases is discussed.