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Microglial cells are critical for glioma growth and progression. However, only little is known about intratumoral microglial behavior and the dynamic interaction with the tumor. Currently the scarce understanding of microglial appearance in malignant gliomas merely originates from histological studies and in vitro investigations. In order to understand the pattern of microglia activity, motility and migration we designed an intravital study in an orthotopic murine glioma model using CX3CR1-eGFP(GFP/wt) mice. We analysed the dynamics of intratumoral microglia accumulation and activity, as well as microglia/tumor blood vessel interaction by epi-illumination and 2-photon laser scanning microscopy. We further investigated cellular and tissue function, including the enzyme activity of intratumoral and microglial NADPH oxidase measured by in vivo fluorescence lifetime imaging. We identified three morphological phenotypes of tumor-associated microglia cells with entirely different motility patterns. We found that NADPH oxidase activation is highly divergent in these microglia subtypes leading to different production levels of reactive oxygen species (ROS). We observed that microglia motility is highest within the perivascular niche, suggesting relevance of microglia/tumor blood vessel interactions. In line, reduction of tumor blood vessels by antivascular therapy confirmed the relevance of the tumor vessel compartment on microglia biology in brain tumors. In summary, we provide new insights into in vivo microglial behavior, regarding both morphology and function, in malignant gliomas. GLIA 2016;64:1210-1226.

Although oligoclonal bands in the cerebrospinal fluid have been a hallmark of multiple sclerosis diagnosis for over three decades, the role of antibody-secreting cells in multiple sclerosis remains unclear. T and B cells are critical for multiple sclerosis pathogenesis, but increasing evidence suggests that plasma cells also contribute, through secretion of autoantibodies. Long-lived plasma cells are known to drive various chronic inflammatory conditions as e.g. systemic lupus erythematosus, however, to what extent they are present in autoimmune central nervous system inflammation has not yet been investigated. In brain biopsies from multiple sclerosis patients and other neurological diseases, we could detect non-proliferating plasma cells (CD138+Ki67−) in the parenchyma. Based on this finding, we hypothesized that long-lived plasma cells can persist in the central nervous system (CNS). In order to test this hypothesis, we adapted the multiple sclerosis mouse model experimental autoimmune encephalomyelitis to generate a B cell memory response. Plasma cells were found in the meninges and the parenchyma of the inflamed spinal cord, surrounded by tissue areas resembling survival niches for these cells, characterized by an up-regulation of chemokines (CXCL12), adhesion molecules (VCAM-1) and survival factors (APRIL and BAFF). In order to determine the lifetime of plasma cells in the chronically inflamed CNS, we labeled the DNA of proliferating cells with 5-ethynyl-2′-deoxyuridine (EdU). Up to five weeks later, we could detect EdU+ long-lived plasma cells in the murine CNS. To our knowledge, this is the first study describing non-proliferating plasma cells directly in the target tissue of a chronic inflammation in humans, as well as the first evidence demonstrating the ability of plasma cells to persist in the CNS, and the ability of the chronically inflamed CNS tissue to promote this persistence. Hence, our results suggest that the CNS provides survival niches for long-lived plasma cells, similar to the niches found in other organs. Targeting these cells in the CNS offers new perspectives for treatment of chronic autoimmune neuroinflammatory diseases, especially in patients who do not respond to conventional therapies. Electronic supplementary material The online version of this article (10.1186/s40478-017-0487-8) contains supplementary material, which is available to authorized users.

Owing to the small size of mitochondria and the complexity of their motility patterns, mitochondrial tracking is technically challenging. Mitochondria are often tracked manually; however, this is time-consuming and prone to measurement error. Here, we examined the suitability of four commercial and open-source software alternatives for automated mitochondrial tracking in neurons compared with manual measurements. We show that all the automated tracking tools dramatically underestimated track length, mitochondrial displacement and movement duration, with reductions ranging from 45 to 77% of the values obtained manually. In contrast, mitochondrial velocity was generally overestimated. Only the number of motile mitochondria and their directionality were similar between strategies. Despite these discrepancies, we show that automated tools successfully detected transport alterations after applying an oxidant agent. Thus, automated methods appear to be suitable for assessing relative transport differences between experimental groups, but not for absolute quantification of mitochondrial dynamics. Although useful for objective and time-efficient measurements of mitochondrial movements, results provided by automated methods should be interpreted with caution.

In human chronic lymphocytic leukemia (CLL) pathogenesis, B-cell antigen receptor signaling seems important for leukemia B-cell ontogeny, whereas the microenvironment influences B-cell activation, tumor cell lodging, and provision of antigenic stimuli. Using the murine Eμ-Tcl1 CLL model, we demonstrate that CXCR5-controlled access to follicular dendritic cells confers proliferative stimuli to leukemia B cells. Intravital imaging revealed a marginal zone B cell–like leukemia cell trafficking route. Murine and human CLL cells reciprocally stimulated resident mesenchymal stromal cells through lymphotoxin–β-receptor activation, resulting in CXCL13 secretion and stromal compartment remodeling. Inhibition of lymphotoxin/lymphotoxin–β-receptor signaling or of CXCR5 signaling retards leukemia progression. Thus, CXCR5 activity links tumor cell homing, shaping a survival niche, and access to localized proliferation stimuli. Significance: CLL and other indolent lymphoma are not curable and usually relapse after treatment, a process in which the tumor microenvironment plays a pivotal role. We dissect the consecutive steps of CXCR5-dependent tumor cell lodging and LTβR-dependent stroma–leukemia cell interaction; moreover, we provide therapeutic solutions to interfere with this reciprocal tumor–stroma cross-talk. Cancer Discov; 4(12); 1448–65. ©2014 AACR. See related commentary by López-Guerra et al., p. 1374 This article is highlighted in the In This Issue feature, p. 1355

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 O−2, 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.

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.

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.

In the bone marrow (BM), memory plasma cells (PCs) survive for long time periods in dedicated microenvironmental survival niches, resting in terms of proliferation. Several cell types, such as eosinophils and reticular stromal cells, have been reported to contribute to the survival niche of memory PCs. However, until now it has not been demonstrated whether the niche is formed by a fixed cellular microenvironment. By intravital microscopy, we provide for the first time evidence that the direct contacts formed between PCs and reticular stromal cells are stable in vivo, and thus the PCs are sessile in their niches. The majority (∼80%) of PCs directly contact reticular stromal cells in a non-random fashion. The mesenchymal reticular stromal cells in contact with memory PCs are not proliferating. On the other hand, we show here that eosinophils in the vicinity of long-lived PCs are vigorously proliferating cells and represent a dynamic component of the survival niche. In contrast, if eosinophils are depleted by irradiation, newly generated eosinophils localize in the vicinity of radiation-resistant PCs and the stromal cells. These results suggest that memory PC niches may provide attraction for eosinophils to maintain stability with fluctuating yet essential accessory cells.

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.

Plasma cells play a crucial role for the humoral immune response as they represent the body's factories for antibody production. The differentiation from a B cell into a plasma cell is controlled by a complex transcriptional network and happens within secondary lymphoid organs. Based on their lifetime, two types of antibody secreting cells can be distinguished: Short-lived plasma cells are located in extrafollicular sites of secondary lymphoid organs such as lymph node medullary cords and the splenic red pulp. A fraction of plasmablasts migrate from secondary lymphoid organs to the bone marrow where they can become long-lived plasma cells. Bone marrow plasma cells reside in special microanatomical environments termed survival niches, which provide factors promoting their longevity. Reticular stromal cells producing the chemokine CXCL12, which is known to attract plasmablasts to the bone marrow but also to promote plasma cell survival, play a crucial role in the maintenance of these niches. In addition, hematopoietic cells are contributing to the niches by providing other soluble survival factors. Here, we review the current knowledge on the factors involved in plasma cell differentiation, their localization and migration. We also give an overview on what is known regarding the maintenance of long lived plasma cells in survival niches of the bone marrow.

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b+ cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an “oxidative stress memory” both in the periphery and CNS compartments, in chronic neuroinflammation. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1497-x) contains supplementary material, which is available to authorized users.

Distribution of mitochondria throughout the cytoplasm is necessary for cellular function and health. Due to their unique, highly polarized morphology, neurons are particularly vulnerable to defects of mitochondrial transport, and its disruption can contribute to neuropathology. In this chapter, we present an ex vivo method for monitoring mitochondrial transport within myelinated sensory and motor axons from spinal nerve roots. This approach can be used to investigate mitochondrial behavior under a number of experimental conditions, e.g., by applying ion channel modulators, ionophores, or toxins, as well as for testing the therapeutic potential of new strategies targeting axonal mitochondrial dynamics.

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.

Background Most patients with common variable immunodeficiency (CVID) present with severely reduced switched memory B-cell counts, and some display an increase of CD21 low B-cell counts (CVID 21low), whereas others do not (CVID 21norm). Altered B-cell receptor (BCR) signaling might contribute to the defective memory formation observed in patients with CVID. Objective We sought to investigate canonical nuclear factor of κ light chain (NF-κB) signaling in B cells from patients with CVID as a central pathway in B-cell differentiation. Methods Degradation of inhibitor of κBα (IκBα) and p65 phosphorylation, nuclear translocation of p65, and regulation of target genes and cell function were investigated after different modes of B-cell stimulation. Results BCR-mediated canonical NF-κB signaling was impaired in all mature naive CVID-derived B cells. This impairment was more profound in naive B cells from CVID 21low patients than CVID 21norm patients and most pronounced in CD21 low B cells. The signaling defect translated into reduced induction of Bcl-xL and IκBα, 2 bona fide target genes of the canonical NF-κB pathway. CD40 ligand– and Toll-like receptor 9–mediated signaling were less strongly altered. Signaling in CD21 low B cells but not CD21 + B cells of patients with HIV was similarly affected. Conclusion Combined with the previous description of disturbed Ca 2+ signaling, the discovery of NF-κB signaling defects, especially in CVID 21low patients, suggests a broad underlying signaling defect affecting especially BCR-derived signals. Given the immune phenotype of monogenic defects affecting Ca 2+ and NF-κB signaling, the latter is more likely to contribute to the humoral deficiency. The strongly disturbed BCR signaling of CD21 low B cells is characteristic for this cell type and independent of the underlying disease.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells - on the level of a few protein molecules in germinal centers.

Oxidative stress and mitochondrial dysfunction appear to contribute to axon degeneration in numerous neurological disorders. However, how these two processes interact to cause axonal damage-and how this damage is initiated-remains unclear. In this study we used transected motor axons from murine peripheral roots to investigate whether oxidative stress alters mitochondrial dynamics in myelinated axons. We show that the nodes of Ranvier are the initial sites of mitochondrial damage induced by oxidative stress. There, mitochondria became depolarized, followed by alterations of the external morphology and disruption of the cristae, along with reduced mitochondrial transport. These mitochondrial changes expanded from the nodes of Ranvier bidirectionally towards both internodes and eventually affected the entire mitochondrial population in the axon. Supplementing axonal bioenergetics by applying nicotinamide adenine dinucleotide and methyl pyruvate, rendered the mitochondria at the nodes of Ranvier resistant to these oxidative stress-induced changes. Importantly, this inhibition of mitochondrial damage protected the axons from degeneration. In conclusion, we present a novel ex vivo approach for monitoring mitochondrial dynamics within axons, which proved suitable for detecting mitochondrial changes upon exogenous application of oxidative stress. Our results indicate that the nodes of Ranvier are the site of initial mitochondrial damage in peripheral axons, and suggest that dysregulation of axonal bioenergetics plays a critical role in oxidative stress-triggered mitochondrial alterations and subsequent axonal injury. These novel insights into the mechanisms underlying axon degeneration may have implications for neurological disorders with a degenerative component.

T cells have an essential role in the induction of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Although for CD4+ T cells it is well established that they contribute to the disease, less is known about the role of CD8+ T cells. Our aim was to determine the individual contribution of CD4+ and CD8+ T cells in myelin oligodendrocyte glycoprotein (MOG)35–55–induced EAE. We investigated MOG35–55–activated CD8+ T cells to clarify their potential to induce or attenuate EAE. We monitored the behavior of CD8+ T cells and their interaction with CD4+ T cells directly at the site of inflammation in the CNS using intravital imaging of the brainstem of EAE-affected living anesthetized mice. We found that mice without CD4+ T cells did not develop relevant clinical signs of disease, although CD8+ T cells were present in the CNS of these mice. These CD8+ T cells displayed reduced motility compared with those in the presence of CD4+ T cells. In mice that harbored CD4+ and CD8+ T cells, we saw a similar extent of clinical signs of EAE as in mice with only CD4+ T cells. Furthermore, the dynamic motility and viability of CD4+ T cells were not disturbed by CD8+ T cells in the lesions of these mice. Therefore, we conclude that in MOG35–55–induced EAE, CD8+ T cell accumulation in the CNS represents instead an epiphenomenon with no impact on clinical disease or on the effects of CD4+ T cells, the latter being the true inducers of the disease.

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.

Background Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated. Methods We applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures. Results By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks. Conclusions The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.

Standard multiphoton laser scanning microscopy (MPLSM) has revolutionized our view of physiologic and pathologic processes in living organisms by enlightening different aspects of cellular choreography in immune responses, that is, cellular motility and co-localization. To understand cellular communication on a molecular level, novel transgenic reporter mice have been generated. In parallel, MPLSM systems have been developed, which make it possible for this technique to be more widely used to address crucial immunological questions. Here, we review the latest progress concerning transgenic mouse technology and multiphoton imaging capacities and discuss further developments which will enable us to visualize all around monitoring and quantification of cellular function at a molecular level directly in vivo.

Initially used mainly in the neurosciences, two-photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two-photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state-of-the-art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge-coupled device (CCD)-based line or field detectors. The new technique's performance is compared with conventional single beam laser-scanning systems that detect signals by means of photomultipliers. We also discuss the use of time- and polarization-resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high-speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungusAspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of3670±140picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

The cofactors NADH and NADPH, hereafter NAD(P)H [NAD(P)= nicotinamide adenine dinucleotide (phosphate)], belong to the principal endogenous indicators of energetic cellular metabolism. Since the metabolic activity of cells is given by the ratio between the concentrations of free and protein-bound NAD(P)H, the development of autofluorescence techniques which accurately measure the modifications to this ratio is particularly significant. Hitherto the methods applied in the monitoring of cellular metabolism have provided either imprecise results, due to interference of the NAD(P)H signal by perturbing factors, or they have required a complicated internal calibration. We employ biexponential fluorescence lifetime imaging (FLIM) in order to discriminate between the free and protein-bound NAD(P)H without any previous calibration. Thus, we have obtained directly, and for the first time, a high-resolution map of cellular metabolism, that is, an image of the contribution of the protein-bound NAD(P)H to the cumulative NAD(P)H fluorescence signal. Moreover, we demonstrate that protein-NAD(P)H complexes characterised by different fluorescence lifetimes are not uniformly distributed all over the cell, as assumed until now, but are concentrated in certain cellular regions. The different fluorescence lifetimes indicate either different protein-NAD(P)H complexes or different bond strengths between NAD(P)H and the protein in these complexes. Since an important aspect in biological applications is to monitor the dynamics of the relevant processes (such as cellular metabolism), rapid dynamical techniques, for example, rapid biexponential fluorescence lifetime imaging, are needed. Furthermore, it is necessary to reduce the evaluation effort as much as possible. Most of the evaluation techniques in multiexponential FLIM are time-expensive iterative methods. The few exceptions are connected with a loss of information, for example, global analysis; or a loss in accuracy, for example, the rapid evaluation technique (RLD). We implement for the first time in FLIM a noniterative, nonrestrictive method originally developed by Prony for approximations of multiexponential decays. The accuracy of this method is verified in biexponential FLIM experiments in time-domain on mixtures of two chromophores both in homogenous and in heterogeneous media. The resulting fluorescence lifetimes agree (within error margins) with the lifetimes of the pure substances determined in monoexponential FLIM experiments. The rapidity of our evaluation method as compared to iterative pixel-by-pixel methods is evidenced by a reduction of the evaluation time by more than one order of magnitude. Furthermore, the applicability of this method for the biosciences is demonstrated in the investigation of cellular metabolism by means of NAD(P)H endogenous fluorescence.

Optic neuritis (ON) is an early syndrome present in patients with multiple sclerosis (MS). It is defined as an autoimmune demyelinating disorder that results in axonal loss and visual disturbances. In order to characterize axonal changes during ON, we used an animal model of MS, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in brown Norway rats. In this model, retinal ganglion cell (RGC) body loss is visible before inflammation of the optic nerve, suggesting that insult to the RGC body might be a primary event that triggers axonal stress, independent of later axonal loss resulting from inflammatory demyelination. This is supported by observations of disturbed axonal ultrastructure prior to demyelination by electron microscopy. In addition, changes in calcium homeostasis during the preclinical phase of the disease are seen which correlate with calpain-mediated cleavage of spectrin, a protein responsible for actin anchoring to the cell membrane and involved in actin remodeling. Therefore, this study aims to determine the timing of disturbances in axonal transport and cytoskeletal integrity, with focus particularly on the preclinical phase of ON. To achieve this, the following techniques have been employed: (1) immunohistochemistry to assess accumulation of various proteins associated with axonal transport (e.g. kinesin and synaptophysin), including the acute axonal injury marker, beta-APP; (2) intravitreal injections of cholera toxin B-FITC, a protein which is retroand anterogradely transported along axons, in order to monitor transport timing; and (3) assays to determine filamentous vs globular actin ratios in the optic nerve, to monitor actin destructuralization. Data obtained so far, indicate that despite evidence of changes in the actin cytoskeletal network and axonal ultrastructure during the preclinical disease stage, the microtubule assembly is not grossly affected, with no detectable perturbations in axonal transport. Instead, axonal transport failure is only seen during the clinical disease phase in the vicinity of demyelinating inflammatory lesions.

To develop new therapeutic strategies for many central nervous system (CNS) diseases, it is essential to observe the motility and function of immune cells within neural tissue. Two-photon laser-scanning microscopy is an outstanding technique for imaging these phenomena under in vivo-like conditions. To gain deeper insight into the pathological phenomena that occur during chronic neuroinflammation of the CNS, we use it to view acute murine hippocampal slices cocultured with different subpopulations of immune cells and to view in vivo the brain stem of anesthetized transgenic mice affected by experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. This protocol describes the preparation of cocultures of acute hippocampal slices with antigen-specific T helper 17 (Th17) cells migrating into the parenchyma, and the preparation of anesthetized mice for imaging the brain stem. We also discuss technical aspects of dual-color, two-photon laser-scanning microscopy that is used to image these samples and that allows for greater flexibility in the choice of fluorophores.

The investigation of physiological phenomena at molecular level within vital cells, for instance metabolic processes, represents a major challenge in life sciences. In this context, measuring methods should be non-invasive for the system under study and should provide high sensitivity and specificity. We demonstrate inhere the power of marker-free, spatially and timely resolved intracellular detection of NAD(P)H-dependent enzymes in the study of phenomena of particular biomedical relevance like phagocytosis or autoimmune reactions. The technique is based on biexponential two-photon laser scanning fluorescence lifetime microscopy (TPLSM FLIM) of the physiologic fluorophore NAD(P)H, which exhibits a fluorescence lifetime τ that is strongly dependent on its chemical environment, i.e. on the enzyme to which NAD(P)H is bound to (free NAD(P)H: τ ∼ 400 ps, enzyme-bound NAD(P)H: τ ∼ 2 ns). In order to verify the feasibility of the method, an in-vitro study with different enzymes in solution was performed. The measured fluorescence lifetimes of enzyme-bound NAD(P)H strongly varied from τ = 960±27 ps for malic dehydrogenase to τ = 3640±101 ps for 3α hydroxysteroid dehydrogenase, which made us confident, that an intracellular selective enzyme detection is possible by means of NAD(P)H-FLIM. We indeed succeeded for the first time to specifically and intracellularly detect the NADPH oxidase, a multi-subunit membrane-bound enzyme complex that catalyzes the reduction of free oxygen to its superoxide anion. Thereby, we selectively monitored the NADPH oxidase during its function within differently activated murine polymorphonuclear leucocytes (PMNs). The experiments revealed a specific fluorescence lifetime of 3670±140 ps for NADPH bound to this enzyme both in humorally activated PMNs, i.e. activation with PMA, and in PMNs phagocytosing the fungus Aspergillus fumigatus, confirming the assembly of NADPH oxidase as highly site-specific.

Understanding the pathological mechanisms in the brain is the decisive step in developing novel specific therapeutic strategies and in curing chronic neuroinflammatory or neurodegenerative diseases like multiple sclerosis (MS). For this purpose, techniques which confer high molecular specificity and spatial resolution, which are non-invasive and allow for dynamics visualization in vivo are necessary. We already showed that NAD(P)H-based fluorescence lifetime imaging (FLIM) is the appropriate technique to detect the activation of NADPH oxidase in murine polymorphonuclear cells and in cells of Nicotiana tabacum during phagocytosis. The fluorescence lifetime of NAD(P)H if bound to NADPH oxidase increases to approx. 3600 ps as compared to the case of NAD(P)H-dependent metabolic enzymes, i.e. approx. 2000 ps. A central yet still not fully understood role in neuronal injury is assigned to microglial NADPH oxidase: this might be a neurotoxic effect due to direct production of reactive oxygen species or, indirectly, due to the induction of NO synthase or a protective role if NADPH oxidase is involved in the phagocytosis of noxious supra-molecular structures like beta-amyloids. By means of NAD(P)H-based FLIM we investigate the activation of NADPH oxidase in murine and human peripheral macropahges as compared to microglia, i.e. macrophages of the central nervous system. The same response to specific chemical activation and to phagocytosis of Stafiloccocus aureus functionalized beads is demonstrated for macrophages and microglia of new-born mice. However, this response is different for microglia of adult, healthy mice as also reflected by their reduced phagocytotic capacity. In pathological context, we investigate the activation of NADPH oxidase in microglia of adult, healthy mice as compared to microglia of mice affected by a murine model of MS. Thus, we were able to identify a new mechanism in chronic neuroinflammation.