Th2 and metabolic responses to nematodes are independent of prolonged host microbiota abrogation.

Antibiotic treatment can lead to elimination of both pathogenic bacteria and beneficial commensals, as well as to altered host immune responses. Here, we investigated the influence of prolonged antibiotic treatment (Abx) on effector, memory and recall Th2 immune responses during the primary infection, memory phase and secondary infection with the small intestinal nematode Heligmosomoides polygyrus. Abx treatment significantly reduced gut bacterial loads, but neither worm burdens, nor worm fecundity in primary infection were affected, only worm burdens in secondary infection were elevated in Abx treated mice. Abx mice displayed trends for elevated effector and memory Th2 responses during primary infection, but overall frequencies of Th2 cells in the siLP, PEC, mLN and in the spleen were similar between Abx treated and untreated groups. Gata3+ effector and memory Th2 cytokine responses also remained unimpaired by prolonged Abx treatment. Similarly, the energy production and defence mechanisms of the host tissue and the parasite depicted by NAD(P)H fluorescence lifetime imaging (FLIM) did not change by the prolonged use of antibiotics. We show evidence that the host Th2 response to intestinal nematodes, as well as host and parasite metabolic pathways are robust and remain unimpaired by host microbiota abrogation.

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range.

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.

Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging.

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 µm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging.

In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes.

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis.

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions.

Aggregation of adult parasitic nematodes in sex-mixed groups analysed by transient anomalous diffusion formalism.

Intestinal parasitic worms are widespread throughout the world, causing chronic infections in humans and animals. However, very little is known about the locomotion of the worms in the host gut. We studied the movement of Heligmosomoides bakeri, naturally infecting mice, and used as an animal model for roundworm infections. We investigated the locomotion of H. bakeri in simplified environments mimicking key physical features of the intestinal lumen, i.e. medium viscosity and intestinal villi topology. We found that the motion sequence of these nematodes is non-periodic, but the migration could be described by transient anomalous diffusion. Aggregation as a result of biased, enhanced-diffusive locomotion of nematodes in sex-mixed groups was detected. This locomotion is probably stimulated by mating and reproduction, while single nematodes move randomly (diffusive). Natural physical obstacles such as high mucus-like viscosity or villi topology slowed down but did not entirely prevent nematode aggregation. Additionally, the mean displacement rate of nematodes in sex-mixed groups of 3.0 × 10-3 mm s-1 in a mucus-like medium is in good agreement with estimates of migration velocities of 10-4 to 10-3 mm s-1 in the gut. Our data indicate H. bakeri motion to be non-periodic and their migration random (diffusive-like), but triggerable by the presence of kin.

Optimized intravital three-photon imaging of intact mouse tibia links plasma cell motility to functional states.

Intravital deep bone marrow imaging is crucial to studying cellular dynamics and functions but remains challenging, and minimally invasive methods are needed. We employed a high pulse-energy 1650 nm laser to perform three-photon microscopy in vivo, reaching ≈400 µm depth in intact mouse tibia. Repetition rates of 3 and 4 MHz allowed us to analyze motility patterns of fast and rare cells within unperturbed marrow and to identify a bi-modal migratory behavior for plasma cells. Third harmonic generation (THG) was identified as a label-free marker for cellular organelles, particularly endoplasmic reticulum, indicating protein synthesis capacity. We found a strong THG signal, suggesting high antibody secretion, in one-third of plasma cells while the rest showed low signals. We discovered an inverse relationship between migratory behavior and THG signal, linking motility to functional plasma cell states. This method may enhance our understanding of marrow microenvironment effects on cellular functions.

Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Center B Cells In Vivo.

Affinity maturation of B cell clones within germinal centers constitutes an important mechanism for immune memory. During this process, B cell receptor signaling capacity is tested in multiple rounds of positive selection. Antigen stimulation and co-stimulatory signals mobilize calcium to switch on gene expression leading to proliferation and survival and to differentiation into memory B cells and plasma cells. Additionally, all these processes require adaption of B cell metabolism, and calcium signaling and metabolic pathways are closely interlinked. Mitochondrial adaption, ROS production, and NADPH oxidase activation are involved in cell fate decisions, but it remains elusive to what extent, especially because the analysis of these dynamic processes in germinal centers has to take place in vivo. Here, we introduce a quantitative intravital imaging method for combined measurement of cytoplasmic calcium concentration and enzymatic fingerprinting in germinal center B cells as a possible tool in order to further examine the relationship of calcium signaling and immunometabolism.

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection.

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.

In vitrovascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture.

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge ofin vitrovascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineeredin vitrotissue constructs.

NAD(P)H fluorescence lifetime imaging of live intestinal nematodes reveals metabolic crosstalk between parasite and host.

Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections.

Method for Multiplexed Dynamic Intravital Multiphoton Imaging.

Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.

Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages.

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.

Teriflunomide Does Not Change Dynamics of Nadph Oxidase Activation and Neuronal Dysfunction During Neuroinflammation.

The multiple sclerosis therapeutic teriflunomide is known to block the de novo synthesis of pyrimidine in mitochondria by inhibiting the enzyme dihydroorotate-dehydrogenase (DHODH). The metabolic processes of oxidative phosphorylation and glycolysis are further possible downstream targets. In healthy adult mice, high levels of dihydroorotate-dehydrogenase (DHODH) activity are measured in the central nervous system (CNS), and DHODH inhibition may cause indirect effects on reactive oxygen species production and NADPH oxidase (NOX) mediated oxidative stress, known to be key aspects of the inflammatory response of the CNS. However, little is known about the effect of teriflunomide on the dynamics of NOX activation in CNS cells and subsequent alterations of neuronal function in vivo. In this study, we employed fluorescence lifetime imaging (FLIM) and phasor analysis of the endogeneous fluorescence of NAD(P)H (nicotinamide adenine dinucleotide phosphate) in the brain stem of mice to visualize the effect of teriflunomide on cellular metabolism. Furthermore, we simultaneously studied neuronal Ca2+ signals in transgenic mice with a FRET-based Troponin C Ca2+ sensor based (CerTN L15) quantified using FRET-FLIM. Hence, we directly correlated neuronal (dys-)function indicated by steadily elevated calcium levels with metabolic activity in neurons and surrounding CNS tissue. Employing our intravital co-registered imaging approach, we could not detect any significant alteration of NOX activation after incubation of the tissue with teriflunomide. Furthermore, we could not detect any changes of the inflammatory induced neuronal dysfunction due to local treatment with teriflunomide. Concerning drug safety, we can confirm that teriflunomide has no metabolic effects on neuronal function in the CNS tissue during neuroinflammation at concentrations expected in orally treated patients. The combined endogenous FLIM and calcium imaging approach developed by us and employed here uniquely meets the need to monitor cellular metabolism as a basic mechanism of tissue functions in vivo.

Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes.

The major function of B lymphocytes is to sense antigens and to produce protective antibodies after activation. This function requires the expression of a B-cell antigen receptor (BCR), and evolutionary conserved mechanisms seem to exist that ensure that B cells without a BCR do not develop nor survive in the periphery. Here, we show that the loss of BCR expression on Burkitt lymphoma cells leads to decreased mitochondrial function and impaired metabolic flexibility. Strikingly, this phenotype does not result from the absence of a classical Syk-dependent BCR signal but rather from compromised ER expansion. We show that the reexpression of immunoglobulins (Ig) in the absence of the BCR signaling subunits Igα and Igß rescues the observed metabolic defects. We demonstrate that immunoglobulin expression is needed to maintain ER homeostasis not only in lymphoma cells but also in resting B cells. Our study provides evidence that the expression of BCR components, which is sensed in the ER and shapes mitochondrial function, represents a novel mechanism of metabolic control in B cells.

Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc.

Analyzing Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Aging and Vascular Amyloid Pathology.

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive [Formula: see text] which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age-dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein.

Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo.

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method.

Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer.

A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4+ T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4+ T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile [Formula: see text] cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent tissue dysfunction is now possible. Hence, the new approach paves the way for deeper insights into the pathology of neuroinflammatory processes on a cellular basis, over the entire disease course.