Probing the immune and healing response of murine ... - OSA Publishing

Probing the immune and healing response of murine intestinal mucosa by time-lapse 2-photon microscopy of laser-induced lesions with realtime dosimetry Regina Orzekowsky-Schroeder,1,5 Antje Klinger,2,5 Sebastian Freidank,1 Norbert Linz,1 Sebastian Eckert,1 Gereon Hüttmann,1 Andreas Gebert,2,4 and Alfred Vogel1,* 1

University of Lübeck, Institute of Biomedical Optics, Peter-Monnik-Weg 4, 23562 Lübeck, Germany 2 University of Lübeck, Institute of Anatomy, Ratzeburger Allee 160, 23538 Lübeck, Germany 4 present address: University of Jena, Institute of Anatomy II, 07740 Jena, Germany 5 Authors have contributed equally * [email protected]

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 laserinduced 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. © 2014 Optical Society of America OCIS codes: (180.4315) Nonlinear microscopy, (170.2520) Fluorescence microscopy, (170.3880) Medical and biological imaging, (350.3390) Laser materials processing, (350.4855) Optical tweezers or optical manipulation, (170.1020) Ablation of tissue, (170.2680) Gastrointestinal, (170.4520) Optical confinement and manipulation.

References and links 1. 2. 3. 4. 5. 6. 7. 8. 9.

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Received 23 Jul 2014; revised 2 Sep 2014; accepted 2 Sep 2014; published 10 Sep 2014

1 October 2014 | Vol. 5, No. 10 | DOI:10.1364/BOE.5.003521 | BIOMEDICAL OPTICS EXPRESS 3521

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1. Introduction The gut constitutes one of the most important barriers between body and environment. The intestinal duct has a mucosal surface of ~200-400 m2 in humans, much larger than the surface of the skin (~2 m2) or lung (~120 m2) [1]. The enormous surface is due to numerous fingerlike projections called villi. The single-layered epithelium of the small bowel holds two complementary functions at the same time: nutrients must be resorbed from the luminal content, and pathogens must be prevented from entering the body. Therefore, the columnar villus epithelium consists of a number of different, highly specialized cell types. While epithelial enterocytes are specialized on resorption, many immune cells such as lymphocytes and antigen-presenting cells reside within the villus epithelium (intraepithelial lymphocytes) or in the lamina propria underneath the epithelium. These cells are continuously moving and active. In case of a local epithelial defect, pathogens might have the chance to enter the lamina propria through that tissue lesion and trigger an immune reaction. Various intestinal disorders

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are associated with repeated damage and impairment of the mucosal surface barrier. This can lead to inflammation, uncontrolled immune response and disequilibrium of the homeostasis. The epithelium of the gastrointestinal tract is a key element of the mucosal barrier, and rapid resealing of the epithelial surface barrier and extrusion of damaged cells into the lumen following injuries are essential to preserve the normal tissue homeostasis. Previous studies investigated the cellular repair process (epithelial restitution) using in vitro or in vivo models [2–5], and Dignass reviewed the molecular regulatory factors that are involved in intestinal epithelial repair [6]. The present intravital study shows, for the first time, intestinal epithelial healing after laser-induced injury on a subcellular and cellular level and in real time. Detailed study of tissue dynamics in response to injury requires the capability of creating lesions at a predetermined location and time with control of the initial lesion size. Moreover, the observational method must provide high resolution and penetration depth as well as the possibility of long-term in vivo experiments under physiological conditions. These prerequisites have made laser nanosurgery in combination with various microscopy modalities the instrument of choice in a number of in vivo studies of biological processes. Laser nanosurgery with femtosecond (fs) laser pulses at MHz repetition rates relies on the creation of low-density plasmas at pulse energies well below the bubble formation threshold. This creates chemical effects mediated by free electrons or multiphoton-absorption that are confined to the focal volume and enable submicrometer precision cuts at arbitrary locations [7]. Examples are the studies of neural network in the brain [8], or the modulation of morphogenetic movements in Drosophila embryos [9]. More energetic laser pulses result in the formation of minute bubbles by thermoelastic stress, and at even higher energies bubble formation is driven by explosive vaporization of the material in the focal volume [7,10]. The bubble formation threshold lies between 10 and 30 nJ, depending on the numerical aperture (NA). Yanik et al. employed amplified femtosecond pulses at 1 kHz repetition rate to study functional regenerations of axons after axotomy [11], and Nishimura et al. used photodisruption of small blood vessels by ultrashort laser pulses to establish models of stroke in rats [12]. Diffraction-limited nanosurgery is also possible with ultraviolet (UV) nanosecond (ns) laser pulses and was used to cut subcellular structures such as the cytoskeleton and to study relaxation processes in cells and developing organisms [13–15]. We discovered recently that minute cavitation bubbles as small as those created by fs pulses can be produced by UV-A ns laser pulses with smooth (Gaussian) temporal shape [16,17]. When single UV-ns laser pulses are used to create well-defined lesions in living tissue, such as murine gut mucosa, the cavitation bubble diameter largely determines the size of the disrupted region [18]. We used this relation to establish an online-dosimetry that monitors the damage size by measuring the size of the laser-produced cavitation bubbles. In previous works we have established an intravital mouse model and shown that autofluorescence based two-photon microscopy is able to reveal the complex morphology and functional dynamics of vital murine intestinal mucosa [19,20]. In this study we combine the mouse model with a compact UV-A laser for nanosurgery and a modality for in vivo online dosimetry of the lesion size based on a probe-beam scattering technique [21]. UV-laser induced lesions are used to examine healing and immune response in the intact intestinal mucosa in living mice and the tissue response is analyzed by two-photon microscopy. Autofluorescence imaging with subcellular resolution enables to easily distinguish individual cells and their cytosolic organelles. We can track the movement of healthy epithelial cells toward the damaged area and of immune cells such as intraepithelial lymphocytes (IELs), polymorphonuclear leucocytes (PMNLs), and antigen-presenting cells (APCs) within the epithelium and lamina propria.

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2. Materials and methods 2.1 Spectrally resolved 2-photon microscopy of laser-induced lesions with real-time dosimetry Figure 1 shows the experimental setup for laser manipulation with online dosimetry and twophoton microscopy of the tissue. Laser manipulation, dosimetry and imaging were done through the same water immersion microscope objective. We used a Zeiss water-immersion 40x C-Apochromat with a numerical aperture (NA) of 1.2. Lesions were produced by focusing single-longitudinal mode (slm) UV laser pulses with 355 nm wavelength, 500 ps pulse duration and Gaussian beam profile (PNV 001525, teem photonics) into the intestinal epithelium. The UV laser beam was coupled into the fluorescence pathway of a two-photon microscope, and expanded to fill the back aperture of the microscope objective. We adjusted the divergence of the UV laser such that the focus falls within the imaging plane of the twophoton microscope. Single pulses for laser nanosurgery were selected from the 10-Hz pulse train using a mechanical shutter. The maximum pulse energy of the UV laser system is 30 µJ and could be attenuated by means of a half-wave plate and polarizer. A fraction of the attenuated beam was directed onto a photodiode. The diode’s signal was used as energy reference and to trigger the oscilloscope. Before each experiment, we measured the pulse energy entering the back entrance pupil of the objective using a photodiode-based energy meter (PD 10, Ophir Optronics Ltd). Data were corrected for the objective’s transmittance (0.43 at 355 nm). For laser surgery, we aimed at a plane 4 µm below the surface. Aiming in axial direction relied on two-photon imaging of the apical cytoplasm of the epithelial cells. In lateral direction we used a cross-hair that indicated the focus position of the UV laser beam.

Fig. 1. Pump-probe setup for UV laser surgery and online dosimetry with spectrally resolved imaging by two-photon fluorescence microscopy. PMT = photomultiplier tube, exp. = beam expander, cw = continuous wave mode, HR = highly reflecting, HT = highly transmitting.

Online-dosimetry of the size of the laser-induced microbubbles was done by a probe-beam scattering technique [10] that was adapted to the in-vivo situation by using a backscattering geometry. A 40 mW slm diode-pumped solid state laser (RCL-040-660-S, CrystaLaser) at

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660 nm wavelength in continuous wave mode (cw) was coupled into the fluorescence pathway of the two-photon microscope and adjusted collinear and confocal with the UV laser beam. Bubble initiation and collapse are marked by the onset and final minimum of the scattering signal. The time difference between initiation and collapse defines the bubble oscillation time, which scales with bubble size [22,23]. For the probe beam detection, we used an interferometric setup with a 50:50 beam splitter and a fast photoreceiver with 200 MHz bandwidth corresponding to < 5 ns temporal resolution (HCA-S-200M-SI, FEMTO Messtechnik GmbH). Interference of the reflections from the bubble’s front and back wall gives rise to amplitude modulations of the scattering signal that allow to track the movement of the bubble wall with nanometer accuracy [21]. Signal evaluation is described in section 3.1. Spectral imaging was performed by means of a commercial two-photon microscope (DermaInspect, Jenlab GmbH) to which a spectral detector with four photomultiplier tubes (PMTs) was added (Hamamatsu R1294A and R1295A). The dichroics were chosen such that tissue autofluorescence is visible in channels 1-3 (380-450 nm, 450-500 nm, 500-580 nm) while red-fluorescent dyes can be detected separately in the fourth channel (580-680 nm). Two-photon excitation was done at 730 nm wavelength with a 80 MHz repetition rate Ti:sapphire laser (MaiTai, Newport Spectra-Physics). The field of view of 150 x 150 µm2 was sampled with 512 x 512 pixels. We showed previously that with this setup intravital imaging of small intestine is possible with subcellular resolution and up to eight hours, and that different cell types in the small intestine can be distinguished based on their morphology and spectral autofluorescence properties [19,20]. 2.2 Mouse model Female Balb/c mice (n = 12), 8 to 10 weeks of age, were purchased from Charles River Laboratories and kept under standard conditions with free access to food and water. Animals were anesthesized by intraperitoneal injection of a combination of Fentanyl (Bayer), Midazolam (Curamed) and Medetomidin (Pfizer). After assuring adequate anesthesia, the mouse was placed on a homoeothermic table for surgery and later experimental handling. Then a tracheostomy was performed and the mouse was connected to a ventilator (Hugo Sachs Elektronik – Harvard Apparatus GmbH). The abdominal cavity was opened and an intestinal loop was gently protruded. The loop was glued onto a heated block with temperature-control and opened lengthwise so as to press the luminal side of the tissue slightly against a fixed microscope coverslip. This procedure minimized movement artifacts due to peristalsis. The mouse on the tempered stage was then placed under the microscope objective as shown in Fig. 2. Figure 2(C) shows a schematic diagram of a segment of an intestinal villus subject to the experimental investigations. During all procedures, the small intestine was constantly moisturized with NaCl (37° C), and core body temperature was maintained at 37° C. The tissue was constantly perfused, as seen by erythrocyte movement

Fig. 2. Setup for the investigation of murine intestinal mucosa in vivo. (A) Anaesthetised balb/c mouse with ventilation and pulse oximetry on a homeothermic table. (B) Schematic drawing of the topology for accessing the intestinal mucosa with the water immersion microscope objective. (C) Schematic diagram of the intestinal mucosa with epithelium and lamina propria at a villus tip (not true to scale).

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phenomena, and cells remained fully motile with no evidence of decreased viability for experiments lasting up to 8 h. During the experiment, the subject’s vital parameters were monitored with a pulse oximeter at the tail vein (MouseOx, Starr Life Sciences) (Fig. 2(A)). The animal experiments were approved by the local government (V742-72241.122). 2.3 Staining protocols and confocal laser scanning microscopy We used propidium iodide (PI) in order to identify damaged cells. A drop of PI solution (1 mg/ml in NaCl) was applied in situ and flushed with Ringer solution after 1-10 min. Because PI is membrane impermeant to healthy cells, it enters exclusively defect cells and intercalates with double-stranded DNA or RNA. When bound to nucleic acids, the fluorescence emission maximum is 617 nm, which falls in the detection range of PMT 4. Immigration of polymorphonuclear leucocytes (PMNL), which belong to the innate immune system, is a possible consequence of laser-induced necrosis. PMNL’s consist of eosinophil and neutrophil granulocytes. To enable identification of these cells, they were marked with antibodies, and reference pictures were taken by confocal microscopy. Their appearance in these pictures was then compared to the 2-photon-microscopic recordings of laser lesions. For confocal microscopy, cryosections (10 µm thick) were fixed in a mixture of methanol and acetone for 10 min at −20° C and transferred to PBS. Monoclonal rabbit anti mouse CCR 3 (Abcam, clone Y31, dilution 1:100), staining eosinophils or monoclonal rat anti-mouse Ly-6G conjugated to Phycoerythrin (BioLegend, clone 1A8), marking neutrophils were incubated for 60 min at room temperature in a moist chamber. Bound primary CCR3 antibody was detected using a goat anti-rabbit IgG antibody conjugated to Alexa Fluor 594. Nuclei were stained using the blue fluorescent dye bisbenzimide Hoechst 33258 (0,1 µg/ml in PBS for 15 min; Sigma). Cryosections were examined using a Zeiss LSM 510 UV Meta confocal laser scanning microscope, equipped with lasers for 364 nm, 488 nm, 543 nm, and 633 nm excitation (Zeiss, Jena, Germany). In addition to fluorescence imaging, a differential interference contrast (DIC) image was recorded. 3. Results and discussion In the following, we first present the dynamics and size of the laser-induced microbubbles as derived from the scattering signals (Figs. 3, 4, 5, and 6). We show that, depending on pulse energy, bubbles are generated in two distinct size regimes (Figs. 7 and 8). We then introduce the physiological situation in healthy murine small intestine as observed by two-photon microscopy as a reference for the tissue response to small lesions (Figs. 9, 10, and 11). Afterwards, we present the immediate tissue effects of microbubbles as well as the healing and immune response to small bubbles of < 2.5 µm radius (Fig. 12) and larger bubbles (Figs. 13, 14, 15, and 16). 3.1 Bubble dynamics and size During optical breakdown, material in the laser focus is ionized and plasma is formed [7,16]. The plasma expansion drives the emission of a shock wave and forms a cavitation bubble [18,24]. The lowest laser pulse energy at which cavitation bubbles are generated, Eth, depends, on the laser pulse duration, wavelength, the numerical aperture used for focusing, and on the local optical properties of the material. In our setup, with NA = 1.2, the threshold energy Eth for bubble formation was 103 nJ in water and 33 nJ in small intestinal mucosa. The reduction of the energy threshold for bubble formation in mucosa is due to the enhanced linear and nonlinear absorption of UV light in biological tissues. Tissues contain many biomolecules possessing energy levels within the band gap of water. Absorption from these excited states may enable a stepwise ionization that reduces the threshold for optical breakdown [25].

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Fig. 3. Scattering signals at the threshold for bubble formation in water (A), and in murine small intestinal epithelium (B). The threshold energies at NA = 1.2 were 103 nJ for water and 33 nJ for intestine, and the bubble oscillation time was Tosc = 29 ns in both cases.

Figure 3 shows scattering signals at the bubble formation thresholds in water and small intestine. The oscillation time, Tosc, of the smallest detectable bubbles was 29 ns in both media. Figure 4 shows typical scattering signals for medium-sized bubbles in water and in epithelium. In water (Fig. 4(A)), the bubble is perfectly spherical, and the signal amplitude is strongly modulated due to alternating constructive and destructive interference of probe-beam light reflected at the bubble front and back walls. The frequency of these interference fringes decreases with decreasing bubble wall velocity during bubble expansion and increases again with increasing velocity during the collapse phase. The bubble expansion time, Texp, corresponds to the time interval between the beginning of the signal and the point at which the fringe frequency is lowest. In analogous fashion the collapse time, Tcoll, is determined. The times for bubble expansion, Texp, and collapse, Tcoll, are almost equal (Tcoll / Texp = 1.05). Scattering signals in epithelial cells as shown in Fig. 4(B) differ markedly from the scattering signals in water. The modulation of the interference fringes is much less pronounced because the bubbles are not perfectly spherical due to local inhomogeneities of the mechanical properties of the cell content that will influence the bubble dynamics. Moreover, the collapse phase takes significantly longer than the bubble expansion (Tcoll / Texp = 2.47), i.e. the signals are asymmetric in time. Finally, the signal amplitude does not recover its original baseline value after the first bubble oscillation but keeps a small positive value and then increases again due to a rebound of the oscillating bubble.

Fig. 4. Interferometric signal from backscattering at a cavitation bubble in water (A), and in small intestine (B). Oscillation times Tosc as well as expansion and collapse times Texp and Tcoll are marked and amount to: (A) Tosc = 356 ns, Tcoll/Texp = 1.04 (B) Tosc = 596 ns, Tcoll/Texp = 2.47. Laser pulse energies were 210 nJ in (A) and 92 nJ in (B).

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Why do bubbles rebound in intestine but not in water? Water has a low viscosity, and cavitation bubbles in water contain mainly water vapour that condenses at the bubble wall during bubble collapse. This leads to a very intense collapse during which a great part of the bubble oscillation energy is dissipated in form of a shock wave [26]. For small bubbles, the collapse is further intensified by surface tension σ which adds a pressure term proportional σ/R. For bubbles with radii smaller than 1 µm the additional pressure becomes larger than the atmospheric pressure. Due to the strong shock wave emission, little energy remains for a rebound after collapse [27]. By contrast, in small intestinal epithelium non-condensable gaseous compounds fill the cavitation bubble. These gaseous compounds are formed during plasma formation due to the dissociation of biomolecules. The collapse is therefore attenuated by the compressibility of this gas. Another attenuating factor is viscous damping of the tissue. Thus, after collapse a small gas-filled residual bubble remains that continues to scatter the probe beam. The bubble rebounds despite the viscosity of the tissue, because only a small fraction of the oscillation energy is converted into an acoustic transient. In water the maximum bubble radius Rmax can be calculated directly from the measured oscillation times [10,22]. In tissue, determination of Rmax from Tosc would lead to erroneous results because the collapse phase is prolonged by viscous damping. However, we shall see that a reasonable value for Rmax can still be obtained based on the bubble expansion time Texp. The elastic forces act symmetrically on bubble expansion and collapse – in a similar fashion as surface tension, which counteracts bubble expansion and accelerates collapse. For the same bubble energy, Tosc is thus shorter and Rmax smaller in an elastic medium than in water. Vice versa, for the same oscillation time, Rmax is larger in an elastic medium than in water. In an elastic medium such as porcine lens, we found Rmax to be 13% larger for a given bubble oscillation time than in water (unpublished data). By contrast, plastic deformation and viscous damping distort the symmetry of the bubble oscillation, mainly by prolonging the collapse phase. In the extreme case of aperiodic damping, Tosc can become infinitely large while Texp still remains finite [28,29]. Therefore, we define a virtual oscillation time T’osc = 2Texp for bubbles in tissue, and calculate Rmax from T’osc as for water. The calculated Rmax values for murine small intestinal epithelium are slightly underestimated due to the use of water parameters in the Gilmore model, because we neglect the elastic forces during expansion. However, the error will be smaller than the above mentioned 13% determined for lens tissue, because mucosa is much softer and behaves more similar to water. By evaluating all interference fringes starting from Rmax obtained from T’osc = 2Texp, we can determine the radius time curve of the bubble oscillation [21] (Figs. 5 and 6). We evaluate the R(t) curve considering that each complete fringe represents a radius change of 1 /4 of the probe laser wavelength λ (660 nm). The bubble walls act as confocal mirrors of an interferometer with pathlength difference Δx of twice the bubble diameter d. Constructive interference occurs if Δx = 2d = 4R = nλ, with n being a natural number. Therefore, one fringe corresponds to a diameter change of λ/2 and a radius change of λ/4. If both interference maxima and minima are evaluated, the R(t) curve is obtained in even smaller steps of λ/8 = 82.5 nm. In the early expansion phase of large bubbles, not all interference fringes can be resolved due to the high velocity of the bubble wall that results in oscillation frequencies at the photodetector which exceed the detector bandwidth. In some cases, this is also true for the late phase of the first bubble collapse. However, fairly often collapse and rebound of bubbles in tissue are so slow that all fringes can be resolved and a continuous R(t) curve is obtained through first and second bubble oscillation (Fig. 6).

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Fig. 5. Scattering signal (A) and radius-time curve (B) of a small cavitation bubble in the epithelium produced with E = 78 nJ. The inset in (A) shows the same signal at a longer time scale. Texp 1 and the endpoints of the first, second and third oscillation are marked with red arrows.

Figure 5 shows the scattering signal (A) and corresponding radius-time curve (B) for a small cavitation bubble produced with E = 78 nJ. During a large part of the first bubble oscillation, the amplitude modulation due to interference is clearly visible. The inset shows the same signal on a longer timescale. Two rebounds were observed as slow increase and decrease of the signal amplitude without detectable interference fringes. We measured Texp 1 = 128 ns, Tosc 1 = 452 ns, i.e. the collapse phase of the first oscillation takes 2.53 times longer than the bubble expansion. The duration of the second and third oscillation lasted significantly longer than the primary oscillation, with Tosc 2 = 529 ns, Tosc 3 = 2.79 µs. The maximum bubble radius is Rmax 1 = 1.79 µm and the residual bubble radius before the first rebound Rmin 1 = 0.63 µm. Figure 6 shows scattering signals and radius-time curves for cavitation bubbles Rmax ≥ 8 µm produced with higher pulse energies of E = 92 nJ (A-C) and E = 103 nJ (D). The signal in 6A, 6B exhibits interference fringes up to the third oscillation. Figure 6(C) shows the radius-time curve up to 6 microseconds after the optical breakdown, and Fig. 6(D) shows a similar curve for another bubble. The R(t) curves in Fig. 6 exhibit a weaker asymmetry during the first oscillation than for the smaller bubbles in Fig. 5. In this respect, the oscillation dynamics start to resemble the bubble dynamics in water. However, the minimum bubble radius during the first collapse is much larger than it would be in water, and the damped oscillation of the R(t) curve converges towards a finite radius value of a long-lasting bubble. This bubble reflects the permanent tissue rupture caused by the rapid initial bubble expansion. For an online dosimetry of laser effects, we do not need to know the entire R(t) curve of the cavitation bubble but can use the maximum bubble radius derived from 2Texp. Figure 7 shows Rmax as a function of pulse energy for single UV laser pulses focussed at NA = 1.2 into murine gut mucosa. Data are compiled from experiments in 12 mice, excluding those scattering signals, for which Texp could not be clearly determined from the interferometric signal. Therefore, data from very small bubbles produced close to threshold, such as in Fig. 3, are missing. We can identify two distinct regimes with different bubble sizes. Regime I contains small bubbles with Rmax = 0.6 µm - 2.4 µm, while regime II contains larger bubbles with Rmax = 7.3 µm – 25.6 µm. The two regimes reflect the two steps of optical breakdown with UV ns laser pulses in transparent dielectrics that have been first observed by Linz [16].

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Fig. 6. Scattering signal in two different time scales (A, B) and radius-time curve (C) of a cavitation bubble in epithelium produced with E = 92 nJ. Texp and Tosc are indicated with arrows. (D) Radius-time curve for a cavitation bubble produced with E = 103 nJ. The inset shows the corresponding scattering signal. (In (C) and (D) only interference maxima were evaluated for the first bubble oscillation, while in the second and third oscillation maxima and minima were used.)

Regime I corresponds to a low density plasma (LDP) while regime II is associated with high-density plasma (HDP) emitting a bright luminescence. For optical breakdown in water, both regimes are separated by a sharp energy threshold [16]. By contrast, in intestinal epithelium the regimes show a certain overlap in terms of energy although a distinct gap is observed between the bubble sizes. This overlap in the energy range of 84-94 nJ is probably due to local inhomogeneities of linear and nonlinear tissue absorption properties. The change of optical breakdown dynamics due to variations of the molecular content in the laser focus highlights the need for an online monitoring of the lesion size. Once a Rmax(E) data set such as in Fig. 7 is available, it can provide a rough estimate of the lesion size for those energy ranges exhibiting little scatter in Rmax, for example Eth < 80 nJ, and Eth > 100 nJ. In the intermediate range 80 nJ < Eth < 100 nJ, interferometric measurements remain necessary. The bubbles in the two regimes differ not only in size but also in their oscillation dynamics. Figure 8 shows the ratio of collapse time and expansion time, Tcoll/Texp, as a function of the expansion time. This ratio measures the asymmetry of the bubble oscillation dynamics owing to the viscoelastic-plastic tissue properties. The asymmetry increases with decreasing bubble size. It is most pronounced in the LDP regime where viscosity has a particularly strong damping effect on bubble collapse, because the influence of viscosity is proportional to 1/R [27,28]. Tcoll/Texp reaches a value of 4.8 for Rmax = 0.6 µm and drops to 1.8 for Rmax = 2.4 µm. With increasing bubble size inertial forces become ever more important, and the oscillation behavior becomes more similar to that of bubbles in water for which expansion and collapse phase are largely symmetric [21].

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Fig. 7. Maximum bubble radius Rmax as a function of pulse energy after application of single UV laser pulses (355 nm, 0.5 ns) focussed with NA 1.2 into intestinal epithelium. Error bars reflect the uncertainty in the determination of Texp from the interferometric signal. Two distinct regimes with different bubble sizes are observed: (I) bubbles with Rmax = 0.6 – 2.4 µm and (II) bubbles with Rmax = 7.3 – 25.6 µm. For comparison, the cartoon in the lower right corner shows the dimensions of an epithelial cell in the small intestine.

Fig. 8. Bubble oscillation asymmetry (collapse time/expansion time) in murine small intestinal mucosa as a function of maximum bubble radius. Error bars for Tcoll/Texp result from the uncertainty in determination of Tosc and Texp from the scattering signals.

Creation of bubbles of different size was used to investigate the tissue response to lesions of different size as will be described in sections 3.3 and 3.4. During each experiment, the bubble radius was determined online. 3.2 Physiological appearance of small intestinal mucosa under two-photon microscopy In this section we show that intravital autofluorescence two-photon microscopy can visualize all tissue elements in the small intestinal mucosa as well as their physiological movements. In our model, normal function of the tissue and cellular dynamics are preserved, because circulatory connections remain intact (Fig. 9, Media 1, Fig. 10, Media 2, Fig. 11, Media 3).

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The luminal side of the small intestine is completely covered by a number of finger-like projections called villi, which enlarge the mucosal surface and thus facilitate absorption of nutrients. Figure 9 shows an image stack through an intestinal villus. The mucosa consists of the epithelium (Fig. 9(A)-9(B)) and underlying supporting loose connective tissue, the so called lamina propria (Fig. 9(C)). The single columnar epithelium consists mostly of enterocytes together with scattered goblet cells, enteroendocrine cells and brush cells [19]. Junctional complexes seal the cells to each other and to the basement membrane. The most important autofluorescent chromophores in the cells are the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) [20] that are located predominantly within the mitochondria and in the cytoplasm. Cellular interfaces consist of nonfluorescent lipids and proteins in the cellular plasma membranes and thus appear as dark lines between the cells. Intraepithelial lymphocytes (IELs) are found in the basal regions between the cells of the epithelium (Fig. 9(B)). Since they consist of mainly a nucleus and only a small rim of cytoplasm they appear as dark structures within the epithelium. They are vigorously moving between the enterocytes [20]. About 50-60 % of the IELS are γδ T cells. These are specialized lymphocytes that ensure the homeostasis of the intestinal epithelium [30] and protect against intestinal inflammation [31,32]. The epithelium rests on the basement membrane, which is not visible in two-photon microscopy, because it consists mainly of type IV collagen that is not autofluorescent [33]. The lamina propria occupies the cores of villi and contains capillaries, lymph vessels, smooth

Fig. 9. Small intestinal mucosa. Stack of optical sections through a villus (Media 1) (A) In 4.9 µm depth, the apical cytoplasm with mitochondrial NAD(P)H exhibits a strong fluorescence signal. Mucus in goblet cells appears dark. (B) In 11.8 µm depth, the image predominantly shows the nuclei of enterocytes (arrowheads) and the nuclei of intraepithelial lymphocytes (IEL; arrows). (C) In 27.9 µm depth, the lamina propria beneath the epithelium is visualized with loose connective tissue containing capillaries (C) and antigen-presenting cells (APC). (D) Schematic diagram of the three different focus planes (A-C). Lymphocytes (L), capillaries (C), antigen-presenting cells (APC). Scale bar 15 µm.

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muscle fibers and lymphoid tissue with antigen-presenting cells and lymphocytes (Fig. 9(C)). Both intraepithelial lymphocytes and lymphoid cells of the lamina propria must elicit robust immunity against harmful pathogens but restrain immune responses against commensal microbes and dietary antigens. The epithelium undergoes physiological turnover every 4-5 days in a process that involves the movement of newly generated cells from the proliferative compartment in the lower crypts to the tip of small intestinal villi [34], presumably by the constant pressure exerted by newly generated cells. About 1400 cells are shed per day from each villus tip, which amounts to ~1011 cells per day from the entire human small intestine [35]. Shedding occurs within minutes and without disruption of epithelial continuity because the surrounding cells constrict the dying cell and meet to form a new multicellular junction below the extruded cell [36–38]. During their lifetime, epithelial cells are anchored to each other and to the basement membrane forming cell-cell and cell-matrix connections. Extracellular matrix molecules involved in these connections provide a constant survival signal to epithelial cells [39]. Aged cells, after travelling from the crypt base to the villus tip die from anoikis, a special form of programmed cell death which is induced by alterations in cell-cell and cell-matrix connections and results in detachment from the basement membrane and extrusion from the cell association [40,41]. The balance between proliferation, differentiation and cell death of the epithelium is critical for normal physiologic function, since excessive cell death might result in barrier defects and, as a consequence, in uncontrolled access of pathogens into the gut wall. The physiological cell shedding process is shown in Fig. 10, Media 2. Cell shedding and mucus production by goblet cells leads to a continuous production of material which is present in the intestinal lumen. Luminal material is transported between intestinal villi due to peristalsis, as visible in Fig. 11, Media 3. Continuation of cell shedding and peristalsis indicates, that normal tissue function is maintained under our experimental conditions. The next sections show how the physiological processes are altered by laser-induced lesions. 3.3 Tissue response to small cavitation bubbles In this section, we discuss the effects of small bubbles with Rmax = 0.6- 2.2 µm (type I bubbles in Fig. 7). Such bubbles are much smaller than the dimensions of a typical epithelial cell with 25-30 µm height and 5-12 µm diameter. Already at the bubble formation threshold, targeted cells appear dark due to a loss of autofluorescence, which is most likely due to destruction of mitochondrial NAD(P)H. Figure 12 shows this type of damage for E = 68 nJ and a bubble radius of 1.9 µm. The corresponding

Fig. 10. Still image (A) and time-lapse video (Media 2) of a physiological cell shedding process in murine small intestinal epithelium. The movie covers 11:50 minutes of observation time, and the sequence is repeated 4 times. The yellow line in (B) indicates the location of the imaging plane.

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Fig. 11. Still image (A) and time-lapse video (Media 3) of material transport between two intestinal villi. The time-lapse covers 2 minutes of observation time and the sequence is repeated 5 times in the movie. The yellow line in (B) indicates the location of the imaging plane.

movie (Media 4) covers the first 20 minutes after the laser pulse. When only single cells were affected by laser surgery, darkening was the only response within the observation period of 30 minutes. The bubble oscillation destroys neither the cell membrane nor junctional complexes to neighboring cells, and basal cell-matrix interactions remain intact. This could be verified by the lack of uptake of propidium iodide (PI), when a PI solution was applied to the mucosal surface. In Fig. 12 and Media 4 one can see that a second cell, which was not hit by the UV laser pulse, is shed into the lumen. The fact that the physiological cell shedding continues to take place even though the villus has been locally damaged by UV laser irradiation indicates that the normal function of the organ is sustained.

Fig. 12. Small intestinal mucosa before (A; A’) and 32 min. after (B, B’) application of a UV laser pulse with E = 68 nJ corresponding to maximum bubble radius of 1.9 µm. The drawing in (A) shows the imaging plane for (A) and (B). (A’) and (B’) are sectional side views from the target cell showing the epithelial layer, basement membrane and underlying lamina propria. (B; B’) Within 32 Minutes after UV laser surgery, the target cell (arrow) loses autofluorescence and turns dark but remains within the epithelium. (A; B) The arrowhead shows an enterocyte undergoing normal cell shedding. A time-lapse movie covers the first 20 minutes after application of the laser pulse (Media 4). Scale bar 15 µm.

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3.4 Tissue response to larger cavitation bubbles When larger bubbles with radii > 7 µm (type II in Fig. 7) are produced in the targeted cell, autofluorescence is lost within the first minute after the laser pulse (Figs. 13-15). Within 30 minutes, the damage has spread and encompasses five (Fig. 13, Media 5) or even ten and more adjacent cells (Fig. 14), which also loose autofluorescence. Since the bubbles are larger than the targeted enterocyte, cell membrane, lateral junctional complexes and basal cellmatrix interactions including the basement membrane are destroyed, leading to necrosis. This was verified by uptake of PI by the cell nucleus shown in Figs. 13 and 15 and in Media 5. Already within one minute after creating the laser lesion, we observed an immune response: polymorphonuclear leucocytes (PMNL), which belong to the innate immune system, accumulated within the area of necrosis (Fig. 14, Media 6 and Fig. 15, Media 7). PMNLs include eosinophil and neutrophil granulocytes that, in the mouse, show multilobular or ring shaped nuclei. They have an average diameter of 12-15 µm and were found to be highly mobile (Media 6). Eosinophil granulocytes are abundant in the small intestinal lamina propria under physiological conditions, which they colonize in a manner dependent on CC-chemokine eotaxin-1 [42] and its receptor CCR3 [43]. Eosinophils are regarded as proinflammatory cells, and their activation status correlates with disease severity [44] but recent work suggests that eosinophils also contribute to the maintenance of tissue integrity [45, 46]. Neutrophil granulocytes are not present in healthy lamina propria but arrive at a site of inflammation due to chemotaxis. After laser damage, they follow chemical signals arising from the lesion site. To confirm that the invading cells observed in Figs. 14, 15 and in Media 6 and Media 7 are indeed PMNLs, Fig. 16 compares PMNLs as seen in the two-photon microscopic images (A, B) with frozen sections of mouse small intestine stained with anti CCR3 for eosinophils (C) and Ly-6G for neutrophils (D). With both imaging modalities, PMNL show multi-lobular and ring shaped nuclei. In two photon microscopy, they furthermore reveal highly fluorescent granules in their cytoplasm, which are likely lysosomes because FAD in lysosomes is a known source for autofluorescence [18]. Both eosinophils and neutrophils are probably attracted by damage-associated molecular patterns (DAMPs) which recruit and activate innate immune cells, aimed at restoration of homeostasis and tissue repair [47,48]. Since DAMPs are produced by necrotic cells, the observation of PMNL after creation of 10-20 µm large bubbles suggests that the laser-induced necrosis triggered the production of DAMPs followed by directed migration of PMNLs into the damaged area. Adenosine triphosphate released by the dying cells further helps

Fig. 13. Small intestinal mucosa after application of a single UV ns laser pulse with E = 98 nJ, corresponding to a maximum bubble radius of 10.2 µm. The drawings show the image plane of (A-E) and (A’-E’). (A-E) Apical cytoplasm of epithelium showing the target cell (1) and four neighbouring cells turning dark within 31 minutes (Media 5). * goblet cells. (A’-E’) Spreading of the damage in the basal part of the epithelium during the first 31 minutes after laser irradiation. In frame E’, after 31 minutes, neighbouring epithelial cells start moving towards the injured area (arrow). Scale bar 10 µm.

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Fig. 14. Small intestinal mucosa before and after application of a single UV ns laser pulse with E = 197 nJ (Media 6). The drawings show at which depth the focal plane is located in each line of images. (A, A’, A”) Healthy villus. (B, B’, B”) One minute after the UV ns laser pulse several cells beside the target cell have lost autofluorescence. The insert in B’ shows a sectional side view of the affected area, showing that the basement membrane (red) is disrupted. (C, C’, C”) 25 minutes after the UV laser pulse the villus has contracted (arrows) and the damage spread further. Necrotic cell material appears in the lumen (arrowheads). The inserts in B”and C” show magnified images of polymorphonuclear leucocytes in the lamina propria. Two cells are marked with * for orientation and serve as fix points to show the growing extent of the lesion. Scale bar 10 µm. The movie shows an image stack through the villus 25 min. after application of the UV laser pulse, corresponding to column C in the figure. Locations of polymorphonuclear leucocytes are indicated with white arrows.

neutrophils attach to the blood vessels walls [49], so that neutrophils can be recruited easily from the venules in the lamina propria and move toward the injured area. The phagocytic function of PMNL probably contributes to the clearance of debris [50], and neutrophils are known to be key cells regulating the switch from proinflammatory to anti-inflammatory conditions [51]. The immediate tissue response of PMNL migration was followed by extrusion of necrotic cells into the lumen and epithelial restitution that becomes visible in the basal part of the epithelium after a few minutes (Fig. 15, Media 7). Healthy epithelial cells adjacent to the injured area migrate into the wound with a measured speed of 0.4 µm/min to cover the

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Fig. 15. Response of small intestinal mucosa after creation of a bubble with Rmax > 7 µm and luminal application of propidium iodide (PI) (Media 7). (A-C) The target cell and two adjacent cells lost autofluorescence and take up PI. (A’-C’) Epithelial restitution (arrows) is seen in the basal part of the epithelium. The insert in C’ is a magnified image section showing a polymorphonuclear leucocyte in the epithelium. The outline of the cell is marked with a white line, and the dark nucleus shows the characteristic complex shape. (D, D’) Two hours after laser exposure, necrotic cells (*) that took up PI are seen above the villus. The insert in D presents a sectional side view of the villus with intact epithelial lining. In the basal part of the epithelium shown in D’ the defect is sealed. Scale bar 20 µm. The movie shows the focus plane in A’-C’ during minutes 3-27 after application of the UV laser pulse, with the sequence being repeated 9 times. Epithelial restitution and a polymorphonuclear lymphocyte are highlighted.

denuded area. These migrating cells form pseudopodia-like structures, reorganize their cytoskeleton, and redifferentiate after closure of the tissue defect [4]. Villus contraction as indicated by the arrows in Fig. 14(C) aids epithelial restitution by effectively reducing the size of the injured surface area to be reepithelialized. Contraction is likely achieved by condensation of microfilaments in cytoplasmic processes of subepithelial myofibroblasts [52]. Epithelial restitution provides a rapid mechanism for covering a defect in the barrier but does not involve proliferation of epithelial cells. After 2 hours at the latest, the large tissue defects were completely healed and the epithelial continuity was established again, resulting in a normal epithelial lining (Fig. 15(D), 15(D’)). However, epithelial cells were flattened and not as columnar as in undamaged epithelium. Epithelial cell proliferation, maturation and differentiation are necessary to replenish the decreased cell pool. The proliferative compartment of epithelial cells is localized in the crypt region, and in normal physiological turnover a gradient of increasingly differentiated epithelial cells is moving along the vertical axis to the villus tip. This process of cell proliferation and differentiation takes about 24 to 96 hours. Therefore, cells damaged by external factors such as laser lesions cannot be replaced by new cells in a few hours after injury, but the process of proliferation and differentiation may be enhanced by regulatory peptides, such as growth factors and cytokines [53,54]. These peptides may also be produced by innate immune cells, such as PMNL invading in the damaged region. When epithelial integrity was established again, PMNL were no longer visible within the epithelium. Apoptosis of PMNL causes specific recognition and clearance by macrophages that starts the process of egress from the injured tissue to the draining lymphatics [55]. Since the necrotic cells were extruded into the lumen, there was no further source of chemo-attractants for PMNL anymore. The fast clearance mechanism of injured cells into the lumen is probably unique to organs such as intestine or lung possessing an interface to the

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Fig. 16. (A, B) Two-photon excited autofluorescence images of polymorphonuclear leucocytes in small intestinal mucosa after application of type II bubbles. Cells show either multilobular (A) or ring shaped nuclei (B). Arrows show highly fluorescent granules in the cytoplasm. (C, D) Merged confocal microscopic images of frozen sections from the small intestine of mice stained with anti CCR 3 for eosinophils (C) and anti Ly-6Gfor neutrophils (D). Like in the two-photon images, the nuclei are either multilobular (C) or ring shaped (D). Scale bar 5 µm.

environment. In other organs, such as heart, brain, kidney or liver, damaged cells remain in situ, since they cannot be removed from the organs and cleared from the body. In such organs, a further inflammatory response is elicited to clear away the injured cells [56–58]. 4. Conclusions We demonstrated that autofluorescence-based two-photon microscopy combined with UV-ns laser-surgery can be used as tool for the intravital investigation of immune and healing response to minute injuries of gut mucosa, which is an important and large interface between body and environment. Microlesions were produced using the disruptive action of cavitation bubbles that are generated by optical breakdown with focussed UV ns laser pulses. The cavitation bubbles’ size and dynamics were monitored online, using a novel probe-beam backscattering technique with < 5 ns temporal and < 80 nm spatial resolution. Bubbles were produced in two size regimes. In regime I, the maximum radius stayed below 2.5 µm, much smaller than the typical size of an enterocyte. Here, the target cell exhibited a loss of autofluorescence but no membrane damage occurred, neighbouring cells were not affected, and no immune response was observed. In regime II, where bubble radii ranged between 7 and 25 µm, the target cell underwent immediate necrosis, and 5-10 neighbouring cells were affected and lost the integrity of their membranes. Within a few minutes after UV laser exposure, polymorphonuclear leucocytes, a type of innate immune cells, migrated into the damaged region and damaged cells were expelled into the intestinal lumen. The lesions healed within 2 hours by stretching and migration of adjacent healthy #217522 - $15.00 USD (C) 2014 OSA



epithelial cells, reestablishing the integrity of the epithelial surface. The expulsion of damaged cells supported by innate immune cells seems to be a characteristic healing response to small lesions of intestinal mucosa. Coverage of the lesion by adjacent cells is possible in healthy tissue but may fail under pathologic conditions where the state and number density of epithelial cells is already compromised and the homeostasis of the epithelium is highly dysregulated. Epithelial homeostasis, that is, the physiological balance of epithelial cell proliferation, differentiation, and apoptosis, is essential for the maintenance of the barrier function. Affections of the barrier functions like inflammatory bowel disease are still not adequately understood and lead to severe tissue damage and massive impairment of concerned patients. This research will serve as a base for future studies, where we will examine how the intestine reacts to microlesions in the case of an existing inflammation and dysregulated homeostasis of the tissue. Our approach may also be useful for the investigation of tissue response to microlesions in other exposed surfaces such as corneal and bronchial epithelia. Comparison between normal and pathological conditions may elucidate tissue response mechanisms relevant for diseases of the cornea (e.g. dry eye) or the respiratory tract (e.g. asthma). Acknowledgements The authors thank Norbert Koop and Björn Martensen for technical assistance with the twophoton microscope setup and infrastructure, and Reinhard Schulz for the design and manufacture of the miniature homoeothermic table. Sarah Kretschmer and Anna Schueth provided valuable assistance in mouse handling. This work was funded by the German Research Foundation (DFG), projects Ge 647/9-1, Hu 629/3-1, and VO 470/14-2.

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