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.

Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.

The basic understanding of inflammatory airway diseases greatly benefits from imaging the cellular dynamics of immune cells. Current imaging approaches focus on labeling specific cells to follow their dynamics but fail to visualize the surrounding tissue. To overcome this problem, we evaluated autofluorescence multiphoton microscopy for following the motion and interaction of cells in the airways in the context of tissue morphology. Freshly isolated murine tracheae from healthy mice and mice with experimental allergic airway inflammation were examined by autofluorescence multiphoton microscopy. In addition, fluorescently labeled ovalbumin and fluorophore-labeled antibodies were applied to visualize antigen uptake and to identify specific cell populations, respectively. The trachea in living mice was imaged to verify that the ex vivo preparation reflects the in vivo situation. Autofluorescence multiphoton microscopy was also tested to examine human tissue from patients in short-term tissue culture. Using autofluorescence, the epithelium, underlying cells, and fibers of the connective tissue, as well as blood vessels, were identified in isolated tracheae. Similar structures were visualized in living mice and in the human airway tissue. In explanted murine airways, mobile cells were localized within the tissue and we could follow their migration, interactions between individual cells, and their phagocytic activity. During allergic airway inflammation, increased number of eosinophil and neutrophil granulocytes were detected that moved within the connective tissue and immediately below the epithelium without damaging the epithelial cells or connective tissues. Contacts between granulocytes were transient lasting 3 min on average. Unexpectedly, prolonged interactions between granulocytes and antigen-uptaking cells were observed lasting for an average of 13 min. Our results indicate that autofluorescence-based imaging can detect previously unknown immune cell interactions in the airways. The method also holds the potential to be used during diagnostic procedures in humans if integrated into a bronchoscope.

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.

Development, alteration and real time dynamics of conjunctiva-associated lymphoid tissue.

PURPOSE: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. METHODS: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. RESULTS: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. CONCLUSIONS: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.