Intravital two-photon microscopy of the native mouse thymus.

The thymus, a key organ in the adaptive immune system, is sensitive to a variety of insults including cytotoxic preconditioning, which leads to atrophy, compression of the blood vascular system, and alterations in hemodynamics. Although the thymus has innate regenerative capabilities, the production of T cells relies on the trafficking of lymphoid progenitors from the bone marrow through the altered thymic blood vascular system. Our understanding of thymic blood vascular hemodynamics is limited due to technical challenges associated with accessing the native thymus in live mice. To overcome this challenge, we developed an intravital two-photon imaging method to visualize the native thymus in vivo and investigated functional changes to the vascular system following sublethal irradiation. We quantified blood flow velocity and shear rate in cortical blood vessels and identified a subtle but significant increase in vessel leakage and diameter ~24 hrs post-sublethal irradiation. Ex vivo whole organ imaging of optically cleared thymus lobes confirmed a disruption of the thymus vascular structure, resulting in an increase in blood vessel diameter and vessel area, and concurrent thymic atrophy. This novel two-photon intravital imaging method enables a new paradigm for directly investigating the thymic microenvironment in vivo.

Objective Analysis of Corneal Nerves and Dendritic Cells. / Objektive Analyse von Hornhautnerven und dendritischen Zellen.

Corneal nerves and dendritic cells are increasingly being visualised to serve as clinical parameters in the diagnosis of ocular surface diseases using intravital confocal microscopy. In this review, different methods of image analysis are presented. The use of deep learning algorithms, which enable automated pattern recognition, is explained in detail using our own developments and compared with other established methods.

Intravital Microscopy for Imaging and Live Cell Tracking of Alveolar Macrophages in Real Time.

Classic in vitro coculture assays of pathogens with host cells have contributed significantly to our understanding of the intracellular lifestyle of several pathogens. Coculture assays with pathogens and eukaryotic cells can be analyzed through various techniques including plating for colony-forming units (CFU), confocal microscopy, and flow cytometry. However, findings from in vitro assays require validation in an in vivo model. Several physiological conditions can influence host-pathogen interactions, which cannot easily be mimicked in vitro. Intravital microscopy (IVM) is emerging as a powerful tool for studying host-pathogen interactions by enabling in vivo imaging of living organisms. As a result, IVM has significantly enhanced the understanding of infection mediated by diverse pathogens. The versatility of IVM has also allowed for the imaging of various organs as sites of local infection. This chapter specifically focuses on IVM conducted on the lung for elucidating pulmonary immune response, primarily involving alveolar macrophages, to pathogens. Additionally, in this chapter we outline the protocol for lung IVM that utilizes a thoracic suction window to stabilize the lung for acquiring stable images.

Quantification of Lymphangiogenesis in the Murine Lymphedema Tail Model Using Intravital Microscopy.

Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated five million Americans. There is no cure for this disease. Assessing lymphatic growth is essential in developing novel therapeutics. Intravital microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous nonviral vector gene delivery using a chip with nanochannel poration in a rapid (<100 ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics in the live mice model. Methods and Results: The murine tail model of lymphedema was utilized. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: TNTpCMV6 group receives pCMV6 (expression vector backbone alone) (n = 6); TNTProx1 group receives pCMV6-Prox1 (n = 6). Lymphatic vessels (fluorescein isothiocyanate [FITC]-dextran stained) and lymphatic branch points (indicating lymphangiogenesis) were analyzed with the confocal/multiphoton microscope. The experimental group TNTProx1 exhibited reduced postsurgical tail lymphedema and increased lymphatic distribution compared to TNTpCMV6 group. More lymphatic branching points (>3-fold) were observed at the TNT site in TNTProx1 group. Conclusions: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. IVM can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

In vivo Labeling and Intravital Imaging of Bacterial Infection using a Near-infrared Fluorescent D-Amino Acid Probe.

Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their in vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for in situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria in vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissue-cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near-infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections in vivo.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in naïve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Diving head-first into brain intravital microscopy.

Tissue microenvironments during physiology and pathology are highly complex, meaning dynamic cellular activities and their interactions cannot be accurately modelled ex vivo or in vitro. In particular, tissue-specific resident cells which may function and behave differently after isolation and the heterogenous vascular beds in various organs highlight the importance of observing such processes in real-time in vivo. This challenge gave rise to intravital microscopy (IVM), which was discovered over two centuries ago. From the very early techniques of low-optical resolution brightfield microscopy, limited to transparent tissues, IVM techniques have significantly evolved in recent years. Combined with improved animal surgical preparations, modern IVM technologies have achieved significantly higher speed of image acquisition and enhanced image resolution which allow for the visualisation of biological activities within a wider variety of tissue beds. These advancements have dramatically expanded our understanding in cell migration and function, especially in organs which are not easily accessible, such as the brain. In this review, we will discuss the application of rodent IVM in neurobiology in health and disease. In particular, we will outline the capability and limitations of emerging technologies, including photoacoustic, two- and three-photon imaging for brain IVM. In addition, we will discuss the use of these technologies in the context of neuroinflammation.

Intravital imaging: dynamic insights into liver immunity in health and disease.

Inflammation is a critical component of most acute and chronic liver diseases. The liver is a unique immunological organ with a dense vascular network, leading to intense crosstalk between tissue-resident immune cells, passenger leucocytes and parenchymal cells. During acute and chronic liver diseases, the multifaceted immune response is involved in disease promoting and repair mechanisms, while upholding core liver immune functions. In recent years, single-cell technologies have unravelled a previously unknown heterogeneity of immune cells, reshaping the complexity of the hepatic immune response. However, inflammation is a dynamic biological process, encompassing various immune cells, orchestrated in temporal and spatial dimensions, and driven by multiorgan signals. Intravital microscopy (IVM) has emerged as a powerful tool to investigate immunity by visualising the dynamic interplay between different immune cells and their surroundings within a near-natural environment. In this review, we summarise the experimental considerations to perform IVM and highlight recent technological developments. Furthermore, we outline the unique contributions of IVM to our understanding of liver immunity. Through the lens of liver disease, we discuss novel immune-mediated disease mechanisms uncovered by imaging-based studies.

Intravital observation of high-scattering and dense-labeling hepatic tissues using multi-photon fluorescence microscopy.

Achieving high-resolution and large-depth microscopic imaging in vivo under conditions characterized by high-scattering and dense-labeling, as commonly encountered in the liver, poses a formidable challenge. Here, through the optimization of multi-photon fluorescence excitation window, tailored to the unique optical properties of the liver, intravital microscopic imaging of hepatocytes and hepatic blood vessels with high spatial resolution was attained. It's worth noting that resolution degradation caused by tissue scattering of excitation light was mitigated by accounting for moderate tissue self-absorption. Leveraging high-quality multi-photon fluorescence microscopy, we discerned structural and functional alterations in hepatocytes during drug-induced acute liver failure. Furthermore, a reduction in indocyanine green metabolism rates associated with acute liver failure was observed using NIR-II fluorescence macroscopic imaging.

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Modeling the Drug delivery Lifecycle of PLG Nanoparticles Using Intravital Microscopy.

Polylactide-co-glycolide (PLG) nanoparticles hold immense promise for cancer therapy due to their enhanced efficacy and biodegradable matrix structure. Understanding their interactions with blood cells and subsequent biodistribution kinetics is crucial for optimizing their therapeutic potential. In this study, three doxorubicin-loaded PLG nanoparticle systems are synthesized and characterized, analyzing their size, zeta potential, morphology, and in vitro release behavior. Employing intravital microscopy in 4T1-tumor-bearing mice, real-time blood and tumor distribution kinetics are investigated. A mechanistic pharmacokinetic model is used to analyze biodistribution kinetics. Additionally, flow cytometry is utilized to identify cells involved in nanoparticle hitchhiking. Following intravenous injection, PLG nanoparticles exhibit an initial burst release (<1 min) and rapidly adsorb to blood cells (<5 min), hindering extravasation. Agglomeration leads to the clearance of one carrier species within 3 min. In stable dispersions, drug release rather than extravasation remains the dominant pathway for drug elimination from circulation. This comprehensive investigation provides valuable insights into the interplay between competing kinetics that influence the lifecycle of PLG nanoparticles post-injection. The findings advance the understanding of nanoparticle behavior and lay the foundation for improved cancer therapy strategies using nanoparticle-based drug delivery systems.

Intravital imaging of the functions of immune cells in the tumor microenvironment during immunotherapy.

Cancer immunotherapy has developed rapidly in recent years and stands as one of the most promising techniques for combating cancer. To develop and optimize cancer immunotherapy, it is crucial to comprehend the interactions between immune cells and tumor cells in the tumor microenvironment (TME). The TME is complex, with the distribution and function of immune cells undergoing dynamic changes. There are several research techniques to study the TME, and intravital imaging emerges as a powerful tool for capturing the spatiotemporal dynamics, especially the movement behavior and the immune function of various immune cells in real physiological state. Intravital imaging has several advantages, such as high spatio-temporal resolution, multicolor, dynamic and 4D detection, making it an invaluable tool for visualizing the dynamic processes in the TME. This review summarizes the workflow for intravital imaging technology, multi-color labeling methods, optical imaging windows, methods of imaging data analysis and the latest research in visualizing the spatio-temporal dynamics and function of immune cells in the TME. It is essential to investigate the role played by immune cells in the tumor immune response through intravital imaging. The review deepens our understanding of the unique contribution of intravital imaging to improve the efficiency of cancer immunotherapy.

Visualizing vasculature and its response to therapy in the tumor microenvironment.

Tumor vasculature plays a critical role in the progression and metastasis of tumors, antitumor immunity, drug delivery, and resistance to therapies. The morphological and functional changes of tumor vasculature in response to therapy take place in a spatiotemporal-dependent manner, which can be predictive of treatment outcomes. Dynamic monitoring of intratumor vasculature contributes to an improved understanding of the mechanisms of action of specific therapies or reasons for treatment failure, leading to therapy optimization. There is a rich history of methods used to image the vasculature. This review describes recent advances in imaging technologies to visualize the tumor vasculature, with a focus on enhanced intravital imaging techniques and tumor window models. We summarize new insights on spatial-temporal vascular responses to various therapies, including changes in vascular perfusion and permeability and immune-vascular crosstalk, obtained from intravital imaging. Finally, we briefly discuss the clinical applications of intravital imaging techniques.

Chronic cranial window implantation for high-resolution intravital imaging of the endothelial glycocalyx in mouse cortex.

The endothelial glycocalyx is an integral component of the brain vascular barrier. Visualizing its structure in vivo is essential to understand its physiological and pathophysiological mechanisms. Here, we present a surgical protocol for chronic cranial window implantation in mice, alongside the use of multiphoton microscopy tools to image the cortical vasculature. We describe steps for cranial window implantation, intravenous injection of fluorescent markers, and intravital imaging. We then detail a technique to quantify glycocalyx thickness using Imaris image analysis software. For complete details on the use and execution of this protocol, please refer to Gray et al. (2023).1.

Intravital Microscopy of Lipopolysaccharide-Induced Inflammatory Changes in Different Organ Systems-A Scoping Review.

Intravital microscopy (IVM) is a powerful imaging tool that captures biological processes in real-time. IVM facilitates the observation of complex cellular interactions in vivo, where ex vivo and in vitro experiments lack the physiological environment. IVM has been used in a multitude of studies under healthy and pathological conditions in different organ systems. IVM has become essential in the characterization of the immune response through visualization of leukocyte-endothelial interactions and subsequent changes within the microcirculation. Lipopolysaccharide (LPS), a common inflammatory trigger, has been used to induce inflammatory changes in various studies utilizing IVM. In this review, we provide an overview of IVM imaging of LPS-induced inflammation in different models, such as the brain, intestines, bladder, and lungs.

Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.

Perfusion Window Chambers Enable Interventional Analyses of Tumor Microenvironments.

Intravital microscopy (IVM) allows spatial and temporal imaging of different cell types in intact live tissue microenvironments. IVM has played a critical role in understanding cancer biology, invasion, metastases, and drug development. One considerable impediment to the field is the inability to interrogate the tumor microenvironment and its communication cascades during disease progression and therapeutic interventions. Here, a new implantable perfusion window chamber (PWC) is described that allows high-fidelity in vivo microscopy, local administration of stains and drugs, and longitudinal sampling of tumor interstitial fluid. This study shows that the new PWC design allows cyclic multiplexed imaging in vivo, imaging of drug action, and sampling of tumor-shed materials. The PWC will be broadly useful as a novel perturbable in vivo system for deciphering biology in complex microenvironments.

Stabilized Window for Intravital Imaging of the Murine Pancreas.

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

Role of in vivo imaging in Head and Neck cancer management.

Intravital microscopy (IVM) and optical coherency tomography (OCT) are powerful optical imaging tools that allow visualization of dynamic biological activities in living subjects with subcellular resolutions. They have been used in preclinical and clinical cancer imaging, providing insights into the complex physiological, cellular, and molecular behaviors of tumors. They have revolutionized cancer diagnosis and therapies, allowing for real-time observation of biologic processes in vivo, including angiogenesis and immune cell interactions. Recent developments in techniques for observing deep tissues of living animals have improved bioluminescent proteins, fluorescent proteins, fluorescent dyes, and detection technologies like two-photon excitation microscopy. These technologies have become indispensable tools in basic sciences, preclinical research, and modern drug development. In Vivo imaging can detect subcellular signaling or metabolic events in living animals, but depth-dependent signal attenuation limits the depth from which significant data can be obtained. Cancer cell motility and invasion are key features of metastatic tumors, but only a small portion of tumor cells are motile and metastasize due to genetic, epigenetic, and microenvironmental heterogeneities.

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.