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.

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.

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.

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.

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.

Two-photon microscopy and fluorescence lifetime imaging of retinal pigment epithelial cells under oxidative stress.

PURPOSE: The aim of this study was to investigate the autofluorescence (AF) of the RPE with two-photon microscopy (TPM) and fluorescence lifetime imaging (FLIM) under normal and oxidative stress conditions. METHODS: Porcine RPE-choroid explants were used for investigation. The RPE-choroid tissue was preserved in a perfusion organ culture system. Oxidative stress was induced by laser photocoagulation with frequency-doubled ND:YAG laser (532 nm) and by exposure to different concentrations (0, 1, 10 mM) of ferrous sulfate (FeSO4) for 1 hour. At indicated time points after exposure, the tissue was examined with TPM and FLIM. Intracellular reactive oxygen species around the photocoagulation lesion were detected with chloromethyl-2'7'-dichlorofluorescein diacetate (CM-H2DCFDA). Melanosomes were isolated from RPE cells and their fluorescence properties were investigated under normal and oxidized conditions. RESULTS: Under normal conditions, AF in RPE cells with TPM is mostly originated from melanosomes, which has a very short fluorescence lifetime (FLT; mean = 117 ps). Under oxidative stress induced by laser irradiation and FeSO4 exposure, bright granular AF appears inside and around RPE cells, whose FLT is significantly longer (mean = 1388 ps) than the FLT of the melanosome-AF. Excitation and emission peaks are found at 710 to 750 nm and 450 to 500 nm, respectively. Oxidative stress increases the fluorescence intensity of the melanosomes but does not change their FLT. CONCLUSIONS: TPM reveals acute oxidative stress-induced bright AF granules inside and around RPE cells which can be clearly discriminated from melanosomes by FLIM. TPM combined with FLIM is a useful tool of live-cell analysis to investigate functional alterations of the RPE.

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.