High-resolution imaging of living gut mucosa: lymphocyte clusters beneath intestinal M cells are highly dynamic structures.

In the Peyer's patches of the small intestine, specialized epithelial cells, the membranous (M) cells, sample antigenic matter from the gut lumen and bring it into contact with cells of the immune system, which are then capable of initiating specific immune reactions. Using autofluorescence 2-photon (A2P) microscopy, we imaged living intestinal mucosa at a 0.5-µm resolution. We identified individual M cells without the aid of a marker and in vivo analyzed their sampling function over hours. Time-lapse recordings revealed that lymphocytes associated with M cells display a remarkable degree of motility with average speed rates of 8.2 µm/min, to form new M cell-associated lymphocyte clusters within less than 15 min. The lymphocytes drastically deform the M cells' cytoplasm and laterally move from one lymphocyte cluster to the next. This implies that the micro-compartment beneath M cells is a highly efficient container to bring potentially harmful antigens into contact with large numbers of immunocompetent cells. Our setup opens a new window for high-resolution 3D imaging of functional processes occurring in lymphoid and mucosal tissues.

Imaging of Wound Closure of Small Epithelial Lesions in the Mouse Trachea.

Integrity of the airway epithelium is essential for normal lung function. However, studies analyzing the repair process of small epithelial lesions in pseudostratified airway epithelium are missing. To follow airway-epithelial wound closure over time, we lesioned small areas of the mouse tracheal epithelium (1 to 12 cells) using a femtosecond laser and followed wound closure up to 6 hours by autofluorescence multiphoton microscopy. Selected lesions were also examined by scanning and transmission electron microscopy and by staining of filamentous actin. Most lesions with a size up to six cells closed by elongation of the surrounding epithelial cells within 6 hours, and all damaged cells were extruded from the epithelium. Electron microscopy confirmed that the surrounding epithelial cells directly closed lesions up to six cells. Most lesions larger than six cells did not close in the observation period of 6 hours, but we observed that basal cells flattened to cover the basement membrane. Delayed wound closure was, in part, attributable to damage of the basement membrane. Cells facing the lesion exhibited increased filamentous actin staining, indicating active cell movement. Not all cells initially facing the lesion participated directly in wound closure, indicating that closure is driven by movement of individual cells rather than a transepithelial coordinated process. Small wounds in the pseudostratified airway epithelium close within hours to preserve epithelial integrity.

Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy.

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions.

Cyclical expression of L-selectin (CD62L) by recirculating T cells.

L-Selectin (CD62L) mediates T-cell entry into lymph nodes. Whether the microenvironment modulates L-selectin expression of T cells during diapedesis and transit is unknown. Therefore, L-selectin expression was determined quantitatively on circulating T cells in blood, lymph nodes and thoracic duct by confocal laser scanning microscopy. We show that in contrast to leukocyte function-associated antigen-1 (CD11a/CD18) and ICAM-1 (CD54), L-selectin expression is cyclically expressed on recirculating T cells. It is reduced to approximately 30% of the blood value during entry across high endothelial venules. Within lymph nodes, CD4(+) T-cell subsets maintain reduced L-selectin expression at a similar level in all compartments (T-cell zone, B-cell zone and medulla). After exit, L-selectin is re-expressed to levels comparable to those of T cells in blood. Apparently, L-selectin levels are not only down-regulated during T-cell activation but also routinely reduced while transmigrating within lymph nodes. L-Selectin down-regulation seems to be ligand independent since it also occurs in the white pulp compartments of the spleen which lack classic L-selectin ligands such as GlyCAM-1 and CD34. In addition, T cells in non-lymphoid organs do not reveal reduced L-selectin levels. Thus, the ability of secondary lymphoid organs to reduce L-selectin expression of T cells prior to activation might be a prerequisite for their characteristic property to induce primary immune responses.

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

Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence-based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions.

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

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.

Data-adaptive image-denoising for detecting and quantifying nanoparticle entry in mucosal tissues through intravital 2-photon microscopy.

Intravital 2-photon microscopy of mucosal membranes across which nanoparticles enter the organism typically generates noisy images. Because the noise results from the random statistics of only very few photons detected per pixel, it cannot be avoided by technical means. Fluorescent nanoparticles contained in the tissue may be represented by a few bright pixels which closely resemble the noise structure. We here present a data-adaptive method for digital denoising of datasets obtained by 2-photon microscopy. The algorithm exploits both local and non-local redundancy of the underlying ground-truth signal to reduce noise. Our approach automatically adapts the strength of noise suppression in a data-adaptive way by using a Bayesian network. The results show that the specific adaption to both signal and noise characteristics improves the preservation of fine structures such as nanoparticles while less artefacts were produced as compared to reference algorithms. Our method is applicable to other imaging modalities as well, provided the specific noise characteristics are known and taken into account.

Carboxypeptidase E modulates intestinal immune homeostasis and protects against experimental colitis in mice.

Enteroendocrine cells (EEC) produce neuropeptides, which are crucially involved in the maintenance of the intestinal barrier. Hence, EEC dysfunction is suggested to be involved in the complex pathophysiology of inflammatory bowel disease (IBD), which is characterized by decreased intestinal barrier function. However, the underlying mechanisms for EEC dysfunction are not clear and suitable models for a better understanding are lacking. Here, we demonstrate that Carboxypeptidase E (CPE) is specifically expressed in EEC of the murine colon and ileum and that its deficiency is associated with reduced intestinal levels of Neuropeptide Y (NPY) and Peptide YY (PYY), which are both produced by EEC. Moreover, cpe-/- mice exhibit an aggravated course of DSS-induced chronic colitis compared to wildtype littermates. In addition, we observed elevated mucosal IL-6 and KC transcript levels already at baseline conditions in cpe-/- mice. Moreover, supernatants obtained from isolated intestinal crypts of cpe-/- mice lead to increased IL-6 and KC expression in MODE-K cells in the presence of LPS. This effect was reversible by co-administration of recombinant NPY, suggesting a CPE mediated immunosuppressive effect in the intestines by influencing the processing of specific neuropeptides. In this context, the chemotaxis of bone marrow derived macrophages towards respective supernatants was enhanced. In conclusion, our data point to an anti-inflammatory role of CPE in the intestine by influencing local cytokine levels and thus regulating the migration of myeloid immune cells into the mucosa. These findings highlight the importance of EEC for intestinal homeostasis and propose EEC as potential therapeutic targets in IBD.

In vivo spectral imaging of different cell types in the small intestine by two-photon excited autofluorescence.

Spectrally resolved two-photon excited autofluorescence imaging is used to distinguish different cell types and functional areas during dynamic processes in the living gut. Excitation and emission spectra of mucosal tissue and tissue components are correlated to spectra of endogenous chromophores. We show that selective excitation with only two different wavelengths within the tuning range of a Ti:sapphire femtosecond laser system yields excellent discrimination between enterocytes, antigen presenting cells and lysosomes based on the excitation and emission properties of their autofluorescence. The method is employed for time-lapse microscopy over up to 8 h. Changes of the spectral signature with the onset of photodamage are demonstrated, and their origin is discussed.