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Intravital microscopy

Hinnerk Schulz-Hildebrandt, Mario Pieper, Charlotte Stehmar, Martin Ahrens, Christian Idel, Barbara Wollenberg, Peter König, Gereon Hüttmann
Intravital microscopy (IVM) offers the opportunity to visualize static and dynamic changes of tissue on a cellular level. It is a valuable tool in research and may considerably improve clinical diagnosis. In contrast to confocal and non-linear microscopy, optical coherence tomography (OCT) with microscopic resolution (mOCT) provides intrinsically cross-sectional imaging. Changing focus position is not needed, which simplifies especially endoscopic imaging. For in-vivo imaging, here we are presenting endo-microscopic OCT (emOCT)...
February 1, 2018: Biomedical Optics Express
P A Stapleton, C R McBride, J Yi, A B Abukabda, T R Nurkiewicz
Preconceptive health encompasses male and female reproductive capability. In females, this takes into account each of the stages of the estrous cycle. Microvascular reactivity varies throughout the estrous cycle in response to hormonal changes and in preparation for pregnancy. Microvascular alterations in response to engineered nanomaterial (ENM) exposure have been described within 24-hours of inhalation; however, the impact upon the uterine vasculature at differing estrous stages and at late-stage pregnancy is unclear...
March 12, 2018: Reproductive Toxicology
Jens Neumann, Sophie Henneberg, Susanne von Kenne, Niklas Nolte, Andreas J Müller, Burkhart Schraven, Michael W Görtler, Klaus G Reymann, Matthias Gunzer, Monika Riek-Burchardt
Inflammation plays an important role in the pathogenesis of ischemic stroke including an acute and prolonged inflammatory process. The role of neutrophil granulocytes as first driver of the immune reaction from the blood site is under debate due to controversial findings. In bone marrow chimeric mice we were able to study the dynamics of tdTomato-expressing neutrophils and GFP-expressing microglia after photothrombosis using intravital two-photon microscopy. We demonstrate the infiltration of neutrophils into the brain parenchyma and confirm a long-lasting contact between neutrophils and microglia as well as an uptake of neutrophils by microglia clearing the brain from peripheral immune cells...
2018: PloS One
Battuya Bayarmagnai, Louisiane Perrin, Kamyar Esmaeili Pourfarhangi, Bojana Gligorijevic
Cancer cell motility and invasion are key features of metastatic tumors. Both are highly linked to tumor microenvironmental parameters, such as collagen architecture or macrophage density. However, due to the genetic, epigenetic and microenvironmental heterogeneities, only a small portion of tumor cells in the primary tumor are motile and furthermore, only a small portion of those will metastasize. This creates a challenge in predicting metastatic fate of single cells based on the phenotype they exhibit in the primary tumor...
2018: Methods in Molecular Biology
Amy F Martinez, Samuel S McCachren, Marianne Lee, Helen A Murphy, Caigang Zhu, Brian T Crouch, Hannah L Martin, Alaattin Erkanli, Narasimhan Rajaram, Kathleen A Ashcraft, Andrew N Fontanella, Mark W Dewhirst, Nirmala Ramanujam
Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice...
March 8, 2018: Scientific Reports
Junichi Kikuta, Masaru Ishii
Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts and osteoblasts, the fundamental process and dynamics have remained elusive. We originally established an advanced imaging system to visualize living bone tissues using intravital two-photon microscopy. By means of this system, we revealed the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in bone tissues...
2018: Clinical Calcium
Monica Manglani, Dorian B McGavern
Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two-photon laser scanning microscopy is a deep tissue imaging technique that provides a means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue...
February 21, 2018: Current Protocols in Immunology
Max Nobis, Sean C Warren, Morghan C Lucas, Kendelle J Murphy, David Herrmann, Paul Timpson
Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology...
March 6, 2018: Journal of Cell Science
Thomas Später, Florian S Frueh, Philipp Karschnia, Michael D Menger, Matthias W Laschke
Dermal substitutes are frequently used for the initial coverage of extensive skin defects. The seeding of these implants with adipose tissue-derived microvascular fragments (ad-MVF) has recently been shown to accelerate their vascularization and incorporation. In the present study we analyzed whether these processes are affected by a thromboprophylactic therapy with the low molecular weight heparin (LMWH) enoxaparin (enox). Green fluorescent protein (GFP)+ ad-MVF were isolated from enox- (8 mg/kg s.c.) and vehicle-treated (0...
March 5, 2018: Wound Repair and Regeneration
Jean-Philippe Desilles, Varouna Syvannarath, Lucas Di Meglio, Célina Ducroux, William Boisseau, Liliane Louedec, Martine Jandrot-Perrus, Jean-Baptiste Michel, Mikael Mazighi, Benoît Ho-Tin-Noé
BACKGROUND: Previous experimental studies have shown that downstream microvascular thromboinflammation is involved in brain damage from acute ischemic stroke. Using intravital microscopy, we investigated and characterized the sequence of downstream microvascular thromboinflammation in an ischemia/reperfusion acute ischemic stroke model. METHODS AND RESULTS: Rats underwent transient monofilament middle cerebral artery (MCA) occlusion. Cerebral microcirculation in the MCA territory was exposed through a craniotomy and analyzed using real-time intravital imaging coupled with laser Doppler interferometry...
March 1, 2018: Journal of the American Heart Association
Andrius Masedunskas, Mark A Appaduray, Christine A Lucas, María Lastra Cagigas, Marco Heydecker, Mira Holliday, Joyce Meiring, Jeff Hook, Anthony Kee, Melissa White, Paul Thomas, Yingfan Zhang, Robert S Adelstein, Tobias Meckel, Till Böcking, Roberto Weigert, Nicole S Bryce, Peter W Gunning, Edna C Hardeman
Many actin filaments in animal cells are co-polymers of actin and tropomyosin. For many of these co-polymers, non-muscle myosin II associates to establish a contractile network. However, the temporal relationship of these 3 proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantitation of the formation of an actin scaffold in real time with the added advantage that it occurs in a living mammal, under physiological conditions...
February 27, 2018: Journal of Cell Science
Melanie Salvermoser, Robert Pick, Ludwig T Weckbach, Annette Zehrer, Phillip Löhr, Maik Drechsler, Markus Sperandio, Oliver Soehnlein, Barbara Walzog
Neutrophil extravasation and interstitial migration are important steps during the recruitment of neutrophils to sites of inflammation. In the present study, we addressed the functional importance of the unconventional class I myosin 1f (Myo1f) for neutrophil trafficking during acute inflammation. In contrast to leukocyte rolling and adhesion, the genetic absence of Myo1f severely compromised neutrophil extravasation into the inflamed mouse cremaster tissue when compared to Myo1f+/+ mice as studied by intravital microscopy...
February 27, 2018: Blood
Ryohei Matsuura, Shigeru Miyagawa, Junichi Kikuta, Masaru Ishii, Yoshiki Sawa
Recent molecular approaches have provided deeper insight on heart failure. However, real-time in vivo cellular dynamics have not been satisfactorily visualized. Here, we present a detailed protocol for in vivo cellular imaging for visualization of the rat heart using two-photon microscopy.
2018: Methods in Molecular Biology
Sayaka Matsumoto, Junichi Kikuta, Masaru Ishii
The liver is a vital organ in the body. It has various essential functions, including detoxification, protein synthesis, and control of infection. Because of its medical importance, liver diseases such as hepatitis and cirrhosis can be crucial for an individual. Exploring dynamics of living cells in the liver would provide the clues for understanding the pathology. However, due to its technical difficulty, few studies have used intravital liver imaging. To resolve this, we have established a novel imaging system for visualizing liver cell dynamics in living animals...
2018: Methods in Molecular Biology
Takashi Hato, Seth Winfree, Pierre C Dagher
Intravital two-photon microscopy is a powerful imaging tool for investigating various biological processes in live animals. This chapter describes an overview of intravital imaging of the rodent kidney including animal surgery, characteristics of renal tubular autofluorescence, in vivo use of fluorescent probes, and renal immune-cell tracking.
2018: Methods in Molecular Biology
Naoto Kawakami
Intravital imaging is a powerful tool for analyzing cellular functions in living animals. In particular, after the two-photon microscopy technique was introduced, a number of studies have visualized important processes. Here, we describe the methods for performing intravital imaging of the central nervous system. This method can be used for imaging not only lymphocytes but also blood vessels for ischemia studies, as well as glia cell activities.
2018: Methods in Molecular Biology
Stefano Sammicheli, Mirela Kuka, Matteo Iannacone
Humoral immune responses depend on B cells encountering antigen (Ag) in lymph nodes (LNs) draining infection sites, getting activated, interacting with different cells, proliferating and differentiating into antibody (Ab)-secreting cells. Each of these events occurs in distinct LN sub-compartments, requiring the migration of B cells from niche to niche in a fast and tightly coordinated fashion. While some of the rules that characterize B cell behavior in secondary lymphoid organs have been elucidated at the population level, we have only limited knowledge of the precise dynamics of B cell interactions with different kinds of LN cells at the single-cell level...
2018: Methods in Molecular Biology
Tomoya Katakai
Live imaging using various microscopic technologies is an indispensable tool for investigating the dynamic nature of immune cells. One of the most powerful techniques is the two-photon laser-scanning microscopy (TP-LSM), which has various advantages in observing deep tissues in vivo. Interstitial T cell migration in the lymph node (LN) is a phenomenon intensively examined using TP-LSM in the field of immunology. Intravital and explant methods have been standards for imaging T cell behaviors in the LN, though there are several limitations...
2018: Methods in Molecular Biology
Yookyung Jung, Joel A Spencer, Anthony P Raphael, Juwell W Wu, Clemens Alt, Judith R Runnels, Briaira Geiger, Charles P Lin
The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins...
2018: Methods in Molecular Biology
Junichi Kikuta, Masaru Ishii
Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts and osteoblasts, the fundamental process and dynamics have remained elusive. To resolve this, we established an intravital bone imaging system using multiphoton microscopy to visualize mature osteoclasts and osteoblasts in living bone.We herein describe the methodology for visualizing the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in living bone tissues using intravital multiphoton microscopy...
2018: Methods in Molecular Biology
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