Probing the immune and healing response of murine intestinal mucosa by time-lapse 2-photon microscopy of laser-induced lesions with real-time dosimetry

Abstract

Gut mucosa is an important interface between body and environment. Immune response and healing processes of murine small intestinal mucosa were investigated by intravital time-lapse two-photon excited autofluorescence microscopy of the response to localized laser-induced damage. Epithelial lesions were created by 355-nm, 500-ps pulses from a microchip laser that produced minute cavitation bubbles. Size and dynamics of these bubbles were monitored using a novel interferometric backscattering technique with 80 nm resolution. Small bubbles (< 2.5 µm maximum radius) merely resulted in autofluorescence loss of the target cell. Larger bubbles (7-25 µm) affected several cells and provoked immigration of immune cells (polymorphonuclear leucocytes). Damaged cells were expelled into the lumen, and the epithelium healed within 2 hours by stretching and migration of adjacent epithelial cells.

Wavelength dependence of femtosecond laser-induced breakdown in water and implications for laser surgery

“…This trend is confirmed by experimental observations. For example, the threshold energy for bubble formation in mouse intestine by 355-nm 500-ps pulses focused at NA = 1.2 amounts to only one third of the respective value in water (33 nJ vs 103 nJ) [130]. Threshold measurements with IR and UV fs laser pulses in water with increasing concentration of bovine serum albumin showed that at a concentration of 10mg/ml, the bubble threshold decreased by 40% for 400 nm pulses, compared to only 7% at 800 nm [57,131].…”

Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation

“…Invasion of PMNLs into the epithelium has also be observed after UV-A laser nanosurgery of intestinal mucosa, involving bubble formation and localized tissue destruction. 29 However, just as during long imaging series with 220 fs laser pulses, no degranulation of PMNLs was observed.…”

“…28 In contrast, PMNLs in the epithelium are only expected under pathological conditions. They are attracted after severe tissue damage, such as that caused by laser-induced cavitation, 29 and can be identified in A2P microcopy by size, speed, and the special shape of their nucleus. 29 PMNLs, as a sign of sterile inflammation, appeared within the epithelium in the long-time imaging series of the basal part of the epithelium (Figs.…”

“…They are attracted after severe tissue damage, such as that caused by laser-induced cavitation, 29 and can be identified in A2P microcopy by size, speed, and the special shape of their nucleus. 29 PMNLs, as a sign of sterile inflammation, appeared within the epithelium in the long-time imaging series of the basal part of the epithelium (Figs. 8, 11, Video 4, Video 5, and Video 6) even at radiant flux values below the threshold of hyperfluorescence formation (corresponding to the green spots in Fig.…”

In Vivo Femtosecond Ablation and Imaging in Bone with a High Repetition Rate Source

“…However, a number of groups have used regeneratively amplified femtosecond pulses to ablate bone, as summarized elsewhere [11]. Additionally, some efforts have targeted a combined approach with imaging-directed ablation in the bone [5,12]. Recently, we have used femtosecond imaging and ablation to enhance cell tracking by bone thinning [13] and to enable the longitudinal evaluation of a bone marrow response to injury [11].…”