Raluca Niesner

Expanding Two-Photon Intravital Microscopy to the Infrared by Means of Optical Parametric Oscillator

Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.

Intravital two-photon microscopy : focus on speed and time resolved imaging modalities

Summary: 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 techniques 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.

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

BackgroundTwo-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.MethodsWe 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.ResultsBy 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.ConclusionsThe 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.

Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm2) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm2) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.

Recent advances in dynamic intravital multi-photon microscopy

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.

Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells

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.

3D-Resolved Investigation of the pH Gradient in Artificial Skin Constructs by Means of Fluorescence Lifetime Imaging

PurposeThe development of substitutes for the human skin, e.g., artificial skin constructs (ASCs), is of particular importance for pharmaceutical and dermatologic research because they represent economical test samples for the validation of new drugs. In this regard, it is essential for the skin substitutes to be reliable models of the genuine skin, i.e., to have similar morphology and functionality. Particularly important is the barrier function, i.e., the selective permeability of the skin, which is strongly related to the epidermal pH gradient. Because the pH significantly influences the permeation profile of ionizable drugs such as nonsteroidal anti-inflammatory drugs, it is of major importance to quantitatively measure the epidermal pH gradient of the ASC and compare it to that of genuine skin.MethodsUsing three-dimensional fluorescence lifetime imaging combined with two-photon scanning microscopy, we measured with submicron resolution the three-dimensional pH gradient in the epidermis of ASCs stained with 2′,7′-bis-(2-carboxyethyl)-5/6-carboxyfluorescein.ResultsSimilar to genuine skin, the surface of the artificial epidermis has an acidic character (pH 5.9), whereas in the deeper layers the pH increases up to 7.0. Moreover, the pH gradient differs in the cell interior (maximally 7.2) and in the intercellular matrix (maximally 6.6). Apart from the similitude of the pH distribution, the genuine and the artificial skin prove to have similar morphologies and to be characterized by similar distributions of the refractive index.ConclusionsArtificial skin is a reliable model of genuine human skin, e.g., in permeability studies, because it is characterized by a similar pH gradient, a similar morphology, and a similar distribution of the refractive index to that of genuine skin.

Time lapse in vivo microscopy reveals distinct dynamics of microglia-tumor environment interactions – a new role for the tumor perivascular space as highway for trafficking microglia

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‐eGFPGFP/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

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

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.

Access to follicular dendritic cells is a pivotal step in murine chronic lymphocytic leukemia B cell activation and proliferation

UNLABELLED 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.