Intravital real-time study of tissue response to controlled laser-induced cavitation using 500-ps UV laser pulses focused in murine gut mucosa under online dosimetry and spectrally resolved 2-photon microscopy

We present a novel experimental setup to intravitally induce and monitor tissue lesions intravitally at a subcellular level in murine small intestinal mucosa. Using single 355-nm, 500-ps laser pulses coupled to a two-photon microscope, we induced optical breakdown with subsequent cavitation bubble formation in the tissue. Imaging was based on spectrally resolved two-photon excited tissue autofluorescence, while online-dosimetry of the induced microbubbles was done by a cw probe-beam scattering technique. From the scattering signal, the bubble size and dynamics could be deduced on a ns time scale. In turn, this signal could be used to control the damage size. This was important for reproducible production of minute effects in the tissue, despite strong biological variations in tissue response to pulsed laser irradiation. After producing local UV damage, cells appeared dark, probably due to destruction of mitochondria and loss of NAD(P)H fluorescence. Within 10 min after cell damage, epithelial cells adjacent to the injured area migrated into the wound to cover the denuded area, resulting in extrusion of the damaged cells from the epithelial layer. Using the nuclear acid stain propidium iodide, we could show that UV pulses induced cell membrane damage with subsequent necrosis, rather than apoptosis. For lesions without disruption of the basement membrane, we did not detect migration of immune cells toward the injured area within observation periods of up to 5 hours. This model will be used in further studies to investigate the intrinsic repair system and immune response to laserinduced lesions of intestinal epithelium in vivo.