Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy
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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.
KeywordsIntravital two-photon microscopy Small intestine Mucosal architecture Cellular migration
The columnar epithelium of the small intestine consists mainly of enterocytes and few scattered goblet cells. Singly dispersed throughout the epithelium are enteroendocrine cells, secreting serotonin and peptide hormones and brush cells that are assumed to represent chemoreceptors of the digestive tract (Hoefer et al. 1996). The study of vital intestinal mucosa has failed in the past, due to the fact that once intestinal cells are removed from basement membrane and underlying stroma, apoptosis is initiated (Hall et al. 1994; Strater et al. 1995). Culture models of intestinal cells, while experimentally tractable, may not convincingly represent the in vivo situation because they generally lack interactions with the supporting tissues, including blood and lymph vessels, nerves and extracellular matrix.
Recent studies, using intravital imaging, even discuss factors uniquely relevant to intact tissue (Miller et al. 2002). These data assume that cell–cell interactions observed in vitro are quite different from those observed in vivo, since lymphatic and circulatory connections are disrupted in explanted preparations, generating unphysiological conditions (Caldwell et al. 2001; Stoll et al. 2002; von Andrian 2002). In this study, we introduce intravital autofluorescence 2-photon (A2P) microscopy based on imaging of naturally occurring endogenous fluorophores such as NAD(P)H and FAD (Denk et al. 1990; Koenig 2000). This novel experimental approach enables the study of the intact intestinal tissue in living mice for up to 8 h, allowing the analysis of cell functions of all participating cells in epithelium and underlying lamina propria. Our approach offers significant advantages for physiological studies of the intestinal mucosa. Using a 40/1.2 water immersion objective, we produce optical sections of the intestinal mucosa at a 0.5-micron resolution, so that individual lysosomes and mitochondria can be easily distinguished. Differentiation of lysosomes and mitochondria was also achieved by fluorescence lifetime imaging microscopy (FLIM), a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image (Skala et al. 2007). Since endocytic and lysosomal pathways play key roles in both the stimulation and implementation of immune responses (Harding et al. 1991; Tulp et al. 1994; Peters et al. 1995; Schmidt et al. 2009), the differentiation of individual cell organelles is necessary to access a complete view of cell composition.
The orchestration of cell migration, cell–cell interactions and intracellular signalling events is fundamental for achieving an accurate view of the physiologically intact intestinal mucosa. A2P microscopy affords an unparalleled view of single-cell spatiotemporal dynamics deep within the intact tissue and we show that individual migrating lymphocytes within the lamina propria can be tracked over several minutes. The application of fluorescent molecular markers, such as UEA-I FITC and organelle-specific dyes complements autofluorescence imaging and delivers additional information on structural characteristics.
The results, presented here not only permit the evaluation of cell physiology and cellular dynamics, but also provide morphological data of most, if not all tissue components in living intestinal mucosa. For the first time, the complex collaboration and organization of all involved cell types of the epithelium and underlying lamina propria is shown. In the present study, we demonstrate that A2P microscopy has the potential of becoming an important tool in multispectral imaging of cellular structure–function relationships and cellular migration in gastrointestinal microscopy. The results presented here open a wide range of interesting and powerful new applications for intravital imaging of the healthy and inflamed intestinal mucosa, evaluating various aspects of cell function, including cell vitality and apoptosis, paracellular permeability and particle transport, endocytosis and blood flow.
Materials and methods
In vivo labeling with fluorescent probes
Hoechst 33258 (Sigma, Schnelldorf, Germany) was dissolved in saline and 200 μg was applied intraperitoneally or intravenously. The lectin Ulex europaeus conjugated to FITC (UEA-I FITC, 0.1 mg/ml, Sigma) was applied luminally.
A2P image analysis
All imaging was conducted using the commercial imaging system JenLab DermaInspect 101 (JenLab, Jena, Germany) equipped with a tunable femtosecond Ti:sapphire laser (pulse width <100 fs, repetition frequency 80–90 MHz; Spectra Physics, Mountain View, CA, USA) using a 40×/1.2 water immersion objective (Zeiss, Jena, Germany). For imaging excitation wavelength was tuned between 710 and 920 nm. A2P imaging was typically done at excitation wavelength of 730 nm, which is known to mainly excite NAD(P)H (Huang et al. 2002) and emission was detected between 380 and 540 nm. Digital images covered a field of 150 × 150 μm at a pixel size of 0.29 × 0.29 μm. Repeated A2P recording of 2D images or 3D image stacks at intervals of 15–30 s resulted in time-lapse series displayed as movies over up to 60 min. Using time correlated single photon counting (TCSPC; Becker and Hickl, Berlin, Germany) we performed fluorescence lifetime imaging. A2P-stacks were processed using IMARIS software (Bitplane, Zurich, Switzerland) for three-dimensional analysis of mucosal morphology.
Lectin labeling and confocal laser scanning microscopy
Cryosections (10-μm thick) were fixed in a mixture of methanol and acetone for 10 min at −20°C and transferred to PBS. The sections were incubated with Ulex europaeus I lectin conjugated to FITC (UEA-I FITC, 0.1 mg/ml, Sigma) for 1 h. Nuclei were stained using Hoechst 33258 (0.1 μg/ml in PBS for 1 h; Sigma). Controls were performed by preincubation of the lectin with fucose overnight. The lectin–gold labeling was performed in PBS containing 1.5% bovine serum albumin-c (BSA-c; Biotrend, Cologne, Germany) and 0.1% sodium azide. Free aldehyde groups were blocked for 15 min in a drop of this buffer (PBS-BSA) containing 0.7% l-lysine. After rinsing in PBS containing 5% bovine serum albumin (Serva; Heidelberg, Germany), 0.1% cold water fish skin gelatin (Biotrend), 1% normal goat serum (Sigma), and 0.05% Tween 20 (Serva), the sections were incubated with PBS-BSA containing the biotinylated Ulex europaeus I lectin at 4°C overnight. After rinsing, the grids were incubated for 4 h with a goat anti-biotin antibody (1:50) conjugated to 20-nm colloidal gold (BioCell; Cardiff, UK). Finally, the grids were treated with 2% glutaraldehyde in PBS, washed in distilled water, and stained with uranium acetate and lead citrate. The sections were examined in a Zeiss EM10 electron microscope (Zeiss; Oberkochen, Germany). Controls were carried out by omitting the lectin and by preincubating the lectin with fucose overnight. Cryosections of lectin labelings were examined using a Zeiss LSM 510 UV meta confocal laser scanning microscope, equipped with lasers for 364, 488, 543, and 633 nm excitation (Zeiss, Jena, Germany). In addition to fluorescence channels, a differential interference contrast (DIC) image was recorded simultaneously.
Transmission electron microscopy
Tissue samples were fixed in a solution of 0.6% paraformaldehyde, 2% glutaraldehyde, and 2 mg/ml CaCl2 in 0.1 M Na–cacodylate–HCl-buffer, pH 7.3, for 16 h. After being rinsed in cacodylate buffer for 30 min, the blocks were postfixed in 2% osmium tetroxide in cacodylate buffer, dehydrated in a graded series of ethanol dilutions, transferred to propylene oxide, and embedded in Araldit (Serva, Heidelberg, Germany) according to standard protocols. Semithin sections, 1 μm in thickness, were stained with toluidine blue. Ultrathin sections, 50–70 nm in thickness, were stained with uranium acetate and lead citrate and examined in a Philips EM400 electron microscope.
In vivo histology of small intestinal epithelium
Three-dimensional architecture of small intestinal mucosa at a subcellular resolution
Lysosomes exhibit different characteristics than mitochondria in A2P microscopy
Fluorescence stains complement autofluorescence imaging in the small intestine
Intravital imaging of motile cells in the lamina propria of small intestinal mucosa
Intravital microscopy is a powerful method for studying fundamental issues of physiology and in vivo morphology. It is currently performed in a variety of organs including brain (Trachtenberg et al. 2002), lymph nodes (Mempel et al. 2004), skin (Masters and So 1999) and kidney (Dunn et al. 2002) and is mostly based on fluorescent dyes introduced to individual cells of interest. Autofluorescence 2-photon (A2P) microscopy, as introduced in this paper, opens a new window which enables time-resolved in vivo histology and physiology of the small intestinal mucosa without the need to add fluorescent stains. Using a high aperture objective (40/1.2), we reach a 0.5-micron resolution so that our approach by far exceeds the resolution of previous intravital studies of the intact intestine (Watson et al. 2005) and vivo imaging is no longer restricted to the cellular level, but extends to subcellular organelle-based imaging of processes such as endocytosis. The greatest advantage of intravital A2P microscopy is the fact that the viability of the tissue is maintained and all tissue elements can be visualized in our experimental setup. The intestine is surgically exposed, but with intact circulatory connections. This was demonstrated by erythrocyte movement phenomena and vigorously moving lymphocytes in the villus lamina propria (Fig. 8, Online Resource ESM_3.mpg). Staining with fluorescent probes and repeated image acquisition did not affect viability (Figs. 6, 7, 8).
A2P microscopy opens a new window for investigating cellular and subcellular dynamics of immune processes within the mucosa of the small intestine. The lamina propria is a loose connective tissue underlying the epithelium and contains numerous immune cells which are considered critical in maintaining the balance between the immune response against harmful pathogens and the induction of tolerance to commensal bacteria and food antigens. Macrophages are the most abundant population of phagocytic cells in the intestine of humans (Lee et al. 1985), demonstrate inflammatory anergy (Smythies et al. 2005) and are assumed to have important functions in maintaining mucosal tolerance. Recent advances have highlighted also a fundamental role for lamina propria dendritic cells, which are highly abundant in rodents, in this function (Uematsu et al. 2008; Varol et al. 2009). Antigen presenting cells (APCs) such as macrophages and dendritic cells give a strong signal in A2P microscopy (Figs. 3, 7, Online Resource, ESM_1.mpg, Online Resource ESM_2.mpg) because they harbor numerous lysosomes, which can easily be distinguished from mitochondria by excitation wavelength and fluorescence lifetime imaging (Fig. 5). Lamina propria lymphocytes (LPL) are also a dominant cell population in the subepithelial tissue and display a phenotype separate from peripheral blood T cells (MacDonald and Pender 1998). Apparently, LPL also play an important role in keeping the immunological homeostasis at the large resorptive interface between the gut lumen and the interior of the body. Our current understanding of immune cell motility and migration in the lamina propria of small intestinal mucosa derives from indirect evidence (Mahida et al. 1997), because it has not been possible to observe these dynamic processes in vivo so far. In the present study, we injected a nuclear dye intraperitoneally 24 h prior to 2-photon microscopy. It is known that lymphatic drainage of the peritoneal cavity takes place toward the mediastinal lymph nodes, prior to systemic dissemination (Marco et al. 1992). We assume that after lymphatic drainage of the dye into the mediastinal lymph nodes, some lymphocytes within the lymph node strongly assimilated the nuclear dye and then migrated into the lamina propria of the small intestine. Morphological aspects and velocities of labeled cells make it most likely that these cells are lymphocytes, which are found to a relatively high proportion in the lamina propria of the small intestinal mucosa and are mostly CD4+, TCR-αβ+ cells (Resendiz-Albor et al. 2004). For the first time, we could analyze the motility of individual labeled lymphocytes within the lamina propria of the small intestine using time-lapse, three dimensional imaging (Fig. 8, Online Resource ESM_3.mpg). The average velocity of 11.8 ± 4.7 μm min−1, with peak velocities that exceeded 24 μm min−1 correlates well with measurements of lymphocytes in intact lymph nodes (Miller et al. 2002). Primary lymphocyte responses require contact-dependent information exchange between lymphocytes and dendritic cells (Mempel et al. 2004). In time series, we observed direct dynamics of lymphocyte–APC interactions (Online Resource ESM_3.mpg), so that applying this technology for visualization of immune cell interactions in intact intestinal mucosa will open a new angle of view on the events involved in antigen-specific immune reactions in the gut. Understanding how immune cells search for pathogens in the intestine is a critical area in which intravital microscopy will probably be the key technology. Creative use of pathogen genetics, mouse genetics and imaging technology, with attention to physiological modes of infection and careful data analysis and modeling, is likely to provide new insights leading to new approaches to combat infections.
The use of A2P microscopy for intravital studies of the intestine, as described in this paper, provides a powerful tool for evaluating tissue histology and cell biology in the most physiologically relevant setting. Our study demonstrates numerous ways how A2P microscopy can be applied to various aspects of intestinal biology and physiology to investigate the complexity and the dynamics of cellular environments with intact circulatory and nervous connections.
This study was supported by the German research foundation (DFG), Projects No. Ge 647/9-1; Ge 647/10-1; HU 629/3-1; HU 629/4-1). We thank H. Manfeldt and C. Örün for excellent technical assistance.
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