In Vivo Two Photon Excited Fluorescence (TPEF) Detection Of Intracellular Microspheres For Cell Tracer Studies In The Cornea

To establish a new methodology using non-linear optical imaging to detect two photon excited fluorescence signals (TPEF) from cells labeled with fluorescent microspheres that were implanted into live rabbit corneas. And also to investigate the clinical application of the Two-photon Lasrer Ophthalmoscope in diagnosis and wound healing process the experiments were designed as follow.Rabbit Corneal fibroblasts (RCF) were labeled by Blue-green fluorescent FluoSpheres (1μm diameter) in culture. Cells were then microinjected into the central cornea of living rabbit cornea. TPEF signals were acquired and cells traced using a newly designed Heidelberg Two-photon Laser Ophthalmoscope at 6 hours and 6 days after injection. RCF labeling and fluorescence specificity were confirmed by laser confocal scanning microscopy. In vitro cell morphology and loss of fluorescent tracer was assessed by inverted epifluorescence microscopy.In vitro cell observations showed tracer up take by RCF after overnight exposure to microspheres. After microinjection into the living rabbit cornea, TPEF signals were detected from labeled cells at 6 hours and 6 days. No fluorescent microspheres were detected under extracellular condition in vivo or in vitro.This study demonstrates the potential of using non-linear optical microscopy to detect TPEF signals from labeled cells microinjected into living tissue. This approach has potential in vivo, long-term applicability to trace live cells without photo-bleaching and toxicity associated with conventional fluorescence and provides deep-penetration and high-resolution .