Intravital Imaging Approaches Research Articles

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope.

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body's outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

A mouse ear skin model to study the dynamics of innate immune responses against the microsporidian Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites related to fungi that cause severe infections in immunocompromised individuals. Encephalitozoon cuniculi is a microsporidian species capable of infecting mammals, including human and rodents. In response to microsporidian infection, innate immune system serves as the first line of defense and allows a partial clearance of the parasite via the innate immune cells, namely macrophages, neutrophils, dendritic cells, and Natural Killer cells. According to the literature, microsporidia bypass this response in vitro by modulating the response of macrophages. In order to study host-parasites interactions in vivo, we developed a model using the mouse ear pinna in combination with an intravital imaging approach. Fluorescent E. cuniculi spores were inoculated into the skin tissue to follow for the first time in real time in an in vivo model the recruitment dynamics of EGFP + phagocytic cells in response to the parasite. The results show that parasites induce an important inflammatory recruitment of phagocytes, with alterations of their motility properties (speed, displacement length, straightness). This cellular response persists in the injection zone, with spores detected inside the phagocytes up to 72 h post-infection. Immunostainings performed on ear tissue cryosections evoke the presence of developing infectious foci from 5 days post-infection, in favor of parasite proliferation in this tissue. Overall, the newly set up mice ear pinna model will increase our understanding of the immunobiology of microsporidia and in particular, to know how they can bypass and hijack the host immune system of an immunocompetent or immunosuppressed host.

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes.

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

Intravital Microscopy in Atherosclerosis Research.

Atherosclerosis is a lipid-driven inflammatory disorder that narrows the arterial lumen and can induce life-threatening complications from coronary artery disease, cerebrovascular disease, and peripheral artery disease. On a mechanistic level, the development of novel cellular-resolution intravital microscopy imaging approaches has recently enabled in vivo studies of underlying biological processes governing disease onset and progress. In particular, multiphoton microscopy has emerged as a promising intravital imaging tool utilizing two-photon-excited fluorescence and second-harmonic generation that provides subcellular resolution and increased imaging depths beyond confocal and epifluorescence microscopy. In this chapter, we describe the state-of-the-art multiphoton microscopy applied to the study of murine atherosclerosis.

Demonstration Of Age-Related Increase In Blood-Brain Barrier Permeability By Longitudinal Intravital Microscopy

Age-related blood-brain barrier disruption and cerebromicrovascular rarefaction contribute importantly to the pathogenesis of both vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). Recent advances in geroscience research enable development of novel interventions to reverse age-related alterations of the cerebral microcirculation for prevention of VCID and AD. To facilitate this research there is an urgent need for sensitive and easy-to-adapt imaging methods, which enable longitudinal assessment of changes in BBB permeability and brain capillarization in aged mice, that could be used in vivo to evaluate treatment efficiency. To enable longitudinal assessment of changes in BBB permeability in aged mice equipped with a chronic cranial window, we adapted and optimized two different intravital two-photon imaging approaches. By assessing relative fluorescence changes over the baseline within a volume of brain tissue, after qualitative image subtraction of the brain microvasculature, we confirmed that in 24 month old C57BL/6J mice cumulative permeability of the microvessels to fluorescent tracers of different molecular weights (0.3 kDa to 40 kDa) is significantly increased as compared to that of 5 month old mice. Real-time recording of vessel cross-sections showed that apparent solute permeability of single microvessels is significantly increased in aged mice vs. young mice. Cortical capillary density, assessed both by intravital two-photon microscopy and optical coherence tomography (OCT) was also decreased in aged mice vs. young mice. The presented methods have been optimized for longitudinal (over the period of 36 weeks) in vivo assessment of cerebromicrovascular health in preclinical geroscience research.

Inhibiting Calcium Release from Ryanodine Receptors Protects Axons after Spinal Cord Injury.

Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to determine axonal fate after spinal cord injury (SCI) in real-time and assess the efficacy of ryanodine receptor inhibition as a potential therapeutic approach to prevent intra-axonal calcium-mediated axonal degeneration. Adult 6-8 week old Thy1YFP transgenic mice that express YFP in axons, as well as triple transgenic Avil-Cre:Ai9:Ai95 mice that express the genetically-encoded calcium indicator GCaMP6f in tdTomato positive axons, were used to visualize axons and calcium changes in axons, respectively. Mice received a mild SCI at the T12 level of the spinal cord. Ryanodine, a RyR antagonist, was given at a concentration of 50 μM intrathecally within 15 min of SCI or delayed 3 h after injury and compared with vehicle-treated mice. RyR inhibition within 15 min of SCI significantly reduced axonal spheroid formation from 1 h to 24 h after SCI and increased axonal survival compared with vehicle controls. Delayed ryanodine treatment increased axonal survival and reduced intra-axonal calcium levels at 24 h after SCI but had no effect on axonal spheroid formation. Together, our results support a role for RyR in secondary axonal degeneration.

Heterogeneity in antiviral B cell responses: Lessons from the movies.

Humoral and cellular responses to viral infections coexist in a dynamic equilibrium that often results in efficient viral clearance. However, in some infections one of the two responses prevails, for instance when an overactivation of cytotoxic T cells is accompanied by weak and insufficient antibody responses. Although the cellular response is usually sufficient to control a primary viral infection, in some cases clearance is not complete and persistent infections ensue. In order to design effective therapeutic or vaccination strategies aiming at inducing early and potent neutralizing antibody responses, a deep knowledge of the cellular and molecular determinants of antiviral immune responses is needed. Here, we review our understanding on the spatiotemporal dynamics of antiviral humoral immune responses, with a particular focus on recent studies using intravital imaging approaches as an insightful complement to more traditional techniques.

Quantitative Pulmonary Neutrophil Dynamics Using Computer-Vision Stabilized Intravital Imaging.

In vivo intravital imaging in animal models in the lung remains challenging owing to respiratory motion artifacts. Here we describe a novel intravital imaging approach based on the computer-vision stabilization algorithm, Computer-Vision Stabilized Intravital Imaging. This method corrects lung movements and deformations at submicron precision in respiring mouse lungs. The precision enables high-throughput quantitative analysis of intravital pulmonary polymorphonuclear neutrophil (PMN) dynamics in lungs. We quantified real-time PMN patrolling dynamics of microvessels in the basal state and PMN recruitment resulting from sequestration in a model of endotoxemia in mice. We focused on determining the marginated pool of PMNs in the lung. Direct visualization of marginated PMNs revealed that they are not static but highly dynamic and undergo repeated cycles of "catch and release." PMNs briefly arrest in larger diameter capillary junction (∼10 μm) and then squeeze into narrower, approximately 5-μm diameter vessels through PMN deformation. We also observed that the sequestered PMNs in lung microvessels lost their migratory capabilities in association with cell morphological change following prolonged endotoxemia. These observations underscore the value of direct visualization and quantitative analysis of PMN dynamics in lungs to study PMN physiology and pathophysiology and role in inflammatory lung injury.

Current Clinical and Pre-Clinical Imaging Approaches to Study the Cancer-Associated Immune System.

In the light of the success and the expected growth of its arsenal, immuno-therapy may become the standard neoadjuvant procedure for many cancers in the near future. However, aspects such as the identity, organization and the activation status of the peri- and intra-tumoral immune cells would represent important elements to weigh in the decision for the appropriate treatment. While important progress in non-invasive imaging of immune cells has been made over the last decades, it falls yet short of entering the clinics, let alone becoming a standard procedure. Here, we provide an overview of the different intra-vital imaging approaches in the clinics and in pre-clinical settings and discuss their benefits and drawbacks for assessing the activity of the immune system, globally and on a cellular level. Stimulated by further research, the future is likely to see many technological advances both on signal detection and emission as well as image specificity and resolution to tackle current hurdles. We anticipate that the ability to precisely determine an immune stage of cancer will capture the attention of the oncologist and will create a change in paradigm for cancer therapy.

New Insights Into Osteoclast Biology.

ABSTRACTOsteoclasts are multinucleated cells that are characterized by their unique ability to resorb large quantities of bone. Therefore, they are frequently the target of therapeutic interventions to ameliorate bone loss. In an adult organism, osteoclasts derive from hematopoietic stem cells and differentiate into osteoclasts within a multistep process under the influence of macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL). Historically, the osteoclast life cycle has been defined as linear, whereby lineage‐committed mononuclear precursors fuse to generate multinucleated highly specialized and localized bone phagocytic cells, which then undergo apoptosis within weeks. Recent advances through lineage tracing, single cell RNA sequencing, parabiosis, and intravital imaging approaches have challenged this dogma, revealing they have greater longevity and the capacity to circulate and undergo cell recycling. Indeed, these new insights highlight that under homeostatic conditions very few incidences of osteoclast apoptosis occur. More importantly, as we revisit the formation and fate of the osteoclast, novel methods to target osteoclast biology in bone pathology and regeneration are emerging. This review briefly summarizes the historical life cycle of osteoclasts and highlights recent discoveries made through advanced methodologies, which have led to a paradigm shift in osteoclast biology. These findings are discussed in light of both existing and emerging bone targeted therapeutics, bone pathologies, and communication between osteoclasts and cells resident in bone or at distant sites. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Intravital Imaging of Candida albicans Identifies Differential In Vitro and In Vivo Filamentation Phenotypes for Transcription Factor Deletion Mutants.

ABSTRACTCandida albicans is an important cause of human fungal infections. A widely studied virulence trait of C. albicans is its ability to undergo filamentation to hyphae and pseudohyphae. Although yeast, pseudohyphae, and hyphae are present in pathological samples of infected mammalian tissue, it has been challenging to characterize the role of regulatory networks and specific genes during in vivo filamentation. In addition, the phenotypic heterogeneity of C. albicans clinical isolates is becoming increasingly recognized, while correlating this heterogeneity with pathogenesis remains an important goal. Here, we describe the use of an intravital imaging approach to characterize C. albicans filamentation in a mammalian model of infection by taking advantage of the translucence of mouse pinna (ears). Using this model, we have found that the in vitro and in vivo filamentation phenotypes of different C. albicans isolates can vary significantly, particularly when in vivo filamentation is compared to solid agar-based assays. We also show that the well-characterized transcriptional regulators Efg1 and Brg1 appear to play important roles both in vivo and in vitro. In contrast, Ume6 is much more important in vitro than in vivo. Finally, strains that are dependent on Bcr1 for in vitro filamentation are able to form filaments in vivo in its absence. This intravital imaging approach provides a new approach to the systematic characterization of this important virulence trait during mammalian infection. Our initial studies provide support for the notion that the regulation and initiation of C. albicans filamentation in vivo is distinct from in vitro induction.IMPORTANCE Candida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection, which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited. We have developed a microscopy method to visualize fluorescently labeled C. albicans undergoing this transition in the subcutaneous tissue of mice. Our studies indicate that the regulation of C. albicans filamentation during infection is distinct from that observed in laboratory conditions.

Demonstration of Age‐Related Increases in Blood‐Brain Barrier Permeability and Microvascular Rarefaction in the Mouse Cerebral Cortex by Longitudinal Intravital Two‐Photon Microscopy and Optical Coherence Tomography (OCT)

Age-related blood-brain barrier disruption and cerebromicrovascular rarefaction contribute importantly to the pathogenesis of both vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). Recent advances in geroscience research enable development of novel interventions to reverse age-related alterations of the cerebral microcirculation for prevention of VCID and AD. To facilitate this research there is an urgent need for sensitive and easy-to-adapt imaging methods, which enable longitudinal assessment of changes in BBB permeability and brain capillarization in aged mice, that could be used in vivo to evaluate treatment efficiency. To enable longitudinal assessment of changes in BBB permeability in aged mice equipped with a chronic cranial window, we adapted and optimized two different intravital two-photon imaging approaches. By assessing relative fluorescence changes over the baseline within a volume of brain tissue, after qualitative image subtraction of the brain microvasculature, we confirmed that in 24 month old C57BL/6J mice cumulative permeability of the microvessels to fluorescent tracers of different molecular weights (0.3 kDa to 40 kDa) is significantly increased as compared to that of 5 month old mice. Real-time recording of vessel cross-sections showed that apparent solute permeability of single microvessels is significantly increased in aged mice vs. young mice. Cortical capillary density, assessed both by intravital two-photon microscopy and optical coherence tomography (OCT) was also decreased in aged mice vs. young mice. The presented methods have been optimized for longitudinal (over the period of 36 weeks) in vivo assessment of cerebromicrovascular health in preclinical geroscience research.

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.

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. Particularly challenging has been the study of dynamic HSC behaviors given that live animal HSC visualization in the native niche still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow (BM)3–5. We find that this subset of LT-HSCs resides in close proximity to both sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in BM niches with the deepest hypoxia and instead are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging reveals that LT-HSCs display limited motility at steady-state. Following activation, LT-HSCs display heterogenous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of BM cavities exhibiting bone-remodeling activities. In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the BM microenvironment, imposed by the stages of bone turnover. Overall, our approach enables direct visualization of HSC behaviors and dissection of heterogeneity in HSC niches.

Dendritic Spines in the Spinal Cord: Live Action Pain.

Dendritic spines are microscopic protrusions on neurons that house the postsynaptic machinery necessary for neurotransmission between neurons. As such, dendritic spine structure is intimately linked with synaptic function. In pathology, dendritic spine behavior and its contribution to disease are not firmly understood. It is well known that dendritic spines are highly dynamic in vivo. In our recent publication, we used an intravital imaging approach, which permitted us to repeatedly visualize the same neurons located in lamina II, a nociceptive processing region of the spinal cord. Using this imaging platform, we analyzed the intravital dynamics of dendritic spine structure before and after nerve injury–induced pain. This effort revealed a time-dependent relationship between the progressive increase in pain outcome, and a switch in the steady-state fluctuations of dendritic spine structure. Collectively, our in vivo study demonstrates how injury that leads to abnormal pain may also contribute to synapse-associated structural remodeling in nociceptive regions of the spinal cord dorsal horn. By combining our live-imaging approach with measures of neuronal activity, such as with the use of calcium or other voltage-sensitive dyes, we expect to gain a more complete picture of the relationship between dendritic spine structure and nociceptive physiology.

Intravital Multiphoton Imaging of the Bone and Bone Marrow Environment.

Over the last two decades, numerous advances in our understanding of bone cell biology and bone mineral homeostasis have been achieved. As a dynamic connective and supportive tissue, bone is constantly sensing and responding to both external mechanical forces and internal systemic and local signals. A variety of intravital imaging approaches have been investigated to identify molecular and cellular processes and to decipher signaling pathways involved in the cellular communication between different types of bone cells that form bone multicellular units. Furthermore, bone multicellular units interact with cells of the immune and hematopoietic system to maintain bone homeostasis. Bone-forming osteoblasts and bone-degrading osteoclasts are situated on the endosteal surface of bone influencing the dynamic remodeling and the regeneration of bone tissue. Osteocytes are found at very unique locations in the bone, closely surrounded by bone matrix, forming a cellular network through their interconnected dendritic processes. Bone marrow cells fill the numerous large cavities inside the bones with various blood cell lineages arising from hematopoietic stem and progenitor cells. A highly complex and interconnected network of arterial vessels and sinusoidal capillaries span through the bone marrow spaces forming an interface between the blood circulation and the bone marrow which allows cell trafficking between both compartments. Live imaging of animals using multiphoton microscopy represents a powerful approach to address the cellular behaviors of bone and bone marrow cells over time and space in their natural tissue microenvironment. The in vivo environment is crucial, because the dynamic behavior of cells is critically influenced by many tissue factors including extracellular components, cytokine and growth factor gradients, and fluid forces, such as blood flow. The review article focuses upon recent advances in multiphoton imaging technologies as well as novel experimental approaches in the understanding of the dynamic molecular and cellular mechanisms underlying bone tissue homeostasis, remodeling, and regeneration under physiological and pathological conditions. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

House dust mites activate nociceptor-mast cell clusters to drive type 2 skin inflammation.

Allergic skin diseases, such as atopic dermatitis (AD), are clinically characterized by severe itching and type 2 immunity-associated hypersensitivity to widely-distributed allergens, including those derived from house dust mites (HDMs). Here we found that HDMs with cysteine-protease activity directly activated peptidergic nociceptors, which are neuropeptide-producing nociceptive sensory neurons, that expressed the ion channel TRPV1 and Tac1, the gene encoding the precursor for the neuropeptide substance P. Intravital imaging and genetic approaches indicated that HDMs-activated nociceptors drove the development of allergic skin inflammation by inducing the degranulation of mast cells contiguous to such nociceptors through the release of substance P and the activation of the cationic molecules receptor MRGPRB2 on mast cells. This data indicates that, after exposure to HDM allergens, activation of TRPV1+Tac1+ nociceptor-MRGPRB2+ sensory clusters represents a key early event in the development of allergic skin reaction.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased invivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

In vivo longitudinal depth-wise visualization of tumorigenesis by needle-shaped side-view confocal endomicroscopy.

In vivo, longitudinal observation of tumorigenesis in a live mouse model over an extended time period has been actively pursued to obtain a better understanding of the cellular and molecular mechanism in a highly complex tumor microenvironment. However, common intravital imaging approaches based on a conventional laser scanning confocal or a two-photon microscope have been mostly limited to the observation of superficial parts of the solid tumor tissue. In this work, we implemented a small diameter needle-shaped side-view confocal endomicroscope that can be directly inserted into a solid tumor in a minimally-invasive manner in vivo. By inserting the side-view endomicroscope into the breast tumor from the surface, we achieved in vivo depth-wise cellular-level visualization of microvasculature and fluorescently labeled tumor cells located deeply inside the tumor. In addition, we successfully performed longitudinal depth-wise visualization of a growing breast tumor over three weeks in a live mouse model, which revealed dynamic changes in microvasculature such as a decreasing amount of intratumoral blood vessels over time.

Capturing the Fantastic Voyage of Monocytes Through Time and Space.

Monocytes are a subset of cells that are categorized together with dendritic cells (DCs) and macrophages in the mononuclear phagocyte system (MPS). Despite sharing several phenotypic and functional characteristics with MPS cells, monocytes are unique cells with the ability to function as both precursor and effector cells in their own right. Before the development of hematopoietic stem cells (HSCs) in utero, monocytes are derived from erythro-myeloid precursors (EMPs) in the fetal liver that are important for populating the majority of tissue resident macrophages. After birth, monocytes arise from bone marrow (BM)-derived HSCs and are released into the circulation upon their maturation, where they survey peripheral tissues and maintain endothelial integrity. Upon sensing of microbial breaches or inflammatory stimuli, monocytes migrate into tissues where their plasticity allows them to differentiate into cells that resemble macrophages or DCs according to the environmental niche. Alternatively, they may also migrate into tissues in the absence of inflammation and remain in an undifferentiated state where they perform homeostatic roles. As monocytes are typically on the move, the availability of intravital imaging approaches has provided further insights into their trafficking patterns in distinct tissue compartments. In this review, we outline the importance of understanding their functional behavior in the context of tissue compartments, and how these studies may contribute towards improved vaccine and future therapeutic strategies.