Intravital Microscopy Evidence That Methylene Blue Should Be a Vasopressor-Sparing Agent in Sepsis Vasoplegia.

Microvasculature failure is expected in sepsis and at higher amine concentrations. Therefore, special attention focused individually on microcirculation is needed. Here, we present that methylene blue can prevent leukocytes from adhering to the endothelium in a rat model of lipopolysaccharide-induced endotoxemia. As hypothesis evidence, an intravital microscopy image is presented.

Context-aware deep learning enables high-efficacy localization of high concentration microbubbles for super-resolution ultrasound localization microscopy.

Ultrasound localization microscopy (ULM) enables deep tissue microvascular imaging by localizing and tracking intravenously injected microbubbles circulating in the bloodstream. However, conventional localization techniques require spatially isolated microbubbles, resulting in prolonged imaging time to obtain detailed microvascular maps. Here, we introduce LOcalization with Context Awareness (LOCA)-ULM, a deep learning-based microbubble simulation and localization pipeline designed to enhance localization performance in high microbubble concentrations. In silico, LOCA-ULM enhanced microbubble detection accuracy to 97.8% and reduced the missing rate to 23.8%, outperforming conventional and deep learning-based localization methods up to 17.4% in accuracy and 37.6% in missing rate reduction. In in vivo rat brain imaging, LOCA-ULM revealed dense cerebrovascular networks and spatially adjacent microvessels undetected by conventional ULM. We further demonstrate the superior localization performance of LOCA-ULM in functional ULM (fULM) where LOCA-ULM significantly increased the functional imaging sensitivity of fULM to hemodynamic responses invoked by whisker stimulations in the rat brain.

Longitudinal intravital imaging of mouse placenta.

Studying placental functions is crucial for understanding pregnancy complications. However, imaging placenta is challenging due to its depth, volume, and motion distortions. In this study, we have developed an implantable placenta window in mice that enables high-resolution photoacoustic and fluorescence imaging of placental development throughout the pregnancy. The placenta window exhibits excellent transparency for light and sound. By combining the placenta window with ultrafast functional photoacoustic microscopy, we were able to investigate the placental development during the entire mouse pregnancy, providing unprecedented spatiotemporal details. Consequently, we examined the acute responses of the placenta to alcohol consumption and cardiac arrest, as well as chronic abnormalities in an inflammation model. We have also observed viral gene delivery at the single-cell level and chemical diffusion through the placenta by using fluorescence imaging. Our results demonstrate that intravital imaging through the placenta window can be a powerful tool for studying placenta functions and understanding the placental origins of adverse pregnancy outcomes.

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM). The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications. This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Intravital imaging of splenic classical monocytes modifying the hepatic CX3CR1+ cells motility to exacerbate liver fibrosis via spleen-liver axis.

CX3CR1+ cells play a crucial role in liver fibrosis progression. However, changes in the migratory behavior and spatial distribution of spleen-derived and hepatic CX3CR1+ cells in the fibrotic liver as well as their influence on the liver fibrosis remain unclear. METHODS: The CX3CR1GFP/+ transgenic mice and CX3CR1-KikGR transgenic mice were used to establish the CCl4-induced liver fibrosis model. Splenectomy, adoptive transfusion of splenocytes, in vivo photoconversion of splenic CX3CR1+ cells and intravital imaging were performed to study the spatial distribution, migration and movement behavior, and regulatory function of CX3CR1+ cells in liver fibrosis. RESULTS: Intravital imaging revealed that the CX3CR1GFP cells accumulated into the fibrotic liver and tended to accumulate towards the central vein (CV) in the hepatic lobules. Two subtypes of hepatic CX3CR1+ cells existed in the fibrotic liver. The first subtype was the interacting CX3CR1GFP cells, most of which were observed to distribute in the liver parenchyma and had a higher process velocity; the second subtype was mobile CX3CR1GFP cells, most of which were present in the hepatic vessels with a faster moving speed. Splenectomy ameliorated liver fibrosis and decreased the number of CX3CR1+ cells in the fibrotic liver. Moreover, splenectomy rearranged CX3CR1GFP cells to the boundary of the hepatic lobule, reduced the process velocity of interacting CX3CR1GFP cells and decreased the number and mobility of mobile CX3CR1GFP cells in the fibrotic liver. Transfusion of spleen-derived classical monocytes increased the process velocity and mobility of hepatic endogenous CX3CR1GFP cells and facilitated liver fibrosis progression via the production of proinflammatory and profibrotic cytokines. The photoconverted splenic CX3CR1+ KikRed+ cells were observed to leave the spleen, accumulate into the fibrotic liver and contact with hepatic CX3CR1+ KikGreen+ cells during hepatic fibrosis. CONCLUSION: The splenic CX3CR1+ monocytes with classical phenotype migrated from the spleen to the fibrotic liver, modifying the migratory behavior of hepatic endogenous CX3CR1GFP cells and exacerbating liver fibrosis via the secretion of cytokines. This study reveals that splenic CX3CR1+ classical monocytes are a key driver of liver fibrosis via the spleen-liver axis and may be potential candidate targets for the treatment of chronic liver fibrosis.

In vivo high-speed microscopy of microbubbles in the chorioallantoic membrane model.

Rationale: The acoustic stimulation of microbubbles within microvessels can elicit a spectrum of therapeutically relevant bioeffects from permeabilization to perfusion shutdown. These bioeffects ultimately arise from complex interactions between microbubbles and microvascular walls, though such interactions are poorly understood particularly at high pressure, due to a paucity of direct in vivo observations. The continued development of focused ultrasound methods hinges in large part on establishing links between microbubble-microvessel interactions, cavitation signals, and bioeffects. Methods: Here, a system was developed to enable simultaneous high-speed intravital imaging and cavitation monitoring of microbubbles in vivo in a chorioallantoic membrane model. Exposures were conducted using the clinical agent DefinityTM under conditions previously associated with microvascular damage (1 MHz, 0.5-3.5 MPa, 5 ms pulse length). Results: Ultrasound-activated microbubbles could be observed and were found to induce localized wall deformations that were more pronounced in smaller microvessels and increased with pressure. A central finding was that microbubbles could extravasate from microvessels (from 34% of vessels at 1 MPa to 79% at 3 MPa) during insonation (94% within 0.5 ms) and that this occurred more frequently and in progressively larger microvessels (up to 180 µm) as pressure was increased. Following microbubble extravasation, transient or sustained red blood cell leakage ensued at the extravasation site in 96% of cases for pressures ≥1 MPa. Conclusions: The results here represent the first high-speed in vivo investigation of high-pressure focused ultrasound-induced microbubble-microvessel interactions. This data provides direct evidence that the process of activated microbubble extravasation can occur in vivo and that it is linked to producing microvessel wall perforations of sufficient size to permit red blood cell leakage. The association of red blood cell leakage with microbubble extravasation provides mechanistic insight into the process of microvessel rupture, which has been widely observed in histology.

More than double the fun with two-photon excitation microscopy.

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Transformer-based spatial-temporal detection of apoptotic cell death in live-cell imaging.

Intravital microscopy has revolutionized live-cell imaging by allowing the study of spatial-temporal cell dynamics in living animals. However, the complexity of the data generated by this technology has limited the development of effective computational tools to identify and quantify cell processes. Amongst them, apoptosis is a crucial form of regulated cell death involved in tissue homeostasis and host defense. Live-cell imaging enabled the study of apoptosis at the cellular level, enhancing our understanding of its spatial-temporal regulation. However, at present, no computational method can deliver robust detection of apoptosis in microscopy timelapses. To overcome this limitation, we developed ADeS, a deep learning-based apoptosis detection system that employs the principle of activity recognition. We trained ADeS on extensive datasets containing more than 10,000 apoptotic instances collected both in vitro and in vivo, achieving a classification accuracy above 98% and outperforming state-of-the-art solutions. ADeS is the first method capable of detecting the location and duration of multiple apoptotic events in full microscopy timelapses, surpassing human performance in the same task. We demonstrated the effectiveness and robustness of ADeS across various imaging modalities, cell types, and staining techniques. Finally, we employed ADeS to quantify cell survival in vitro and tissue damage in mice, demonstrating its potential application in toxicity assays, treatment evaluation, and inflammatory dynamics. Our findings suggest that ADeS is a valuable tool for the accurate detection and quantification of apoptosis in live-cell imaging and, in particular, intravital microscopy data, providing insights into the complex spatial-temporal regulation of this process.

Real-Time Multiphoton Intravital Microscopy of Drug Extravasation in Tumours during Acoustic Cluster Therapy.

Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5-10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT's mechanisms and potential in localised drug delivery.

Investigating clot-flow interactions by integrating intravital imaging with in silico modeling for analysis of flow, transport, and hemodynamic forces.

As a blood clot forms, grows, deforms, and embolizes following a vascular injury, local clot-flow interactions lead to a highly dynamic flow environment. The local flow influences transport of biochemical species relevant for clotting, and determines the forces on the clot that in turn lead to clot deformation and embolization. Despite this central role, quantitative characterization of this dynamic clot-flow interaction and flow environment in the clot neighborhood remains a major challenge. Here, we propose an approach that integrates dynamic intravital imaging with computer geometric modeling and computational flow and transport modeling to develop a unified in silico framework to quantify the dynamic clot-flow interactions. We outline the development of the methodology referred to as Intravital Integrated In Silico Modeling or IVISim, and then demonstrate the method on a sample set of simulations comprising clot formation following laser injury in two mouse cremaster arteriole injury model data: one wild-type mouse case, and one diYF knockout mouse case. Simulation predictions are verified against experimental observations of transport of caged fluorescent Albumin (cAlb) in both models. Through these simulations, we illustrate how the IVISim methodology can provide insights into hemostatic processes, the role of flow and clot-flow interactions, and enable further investigations comparing and contrasting different biological model scenarios and parameter variations.

Cancer Imaging by Intravital Microscopy: The Dorsal Window Chamber Model.

Intravital microscopy allows a direct visualization of cells' behavior in their environment in a living organism with all its complexity. With appropriated models, longitudinal studies of structural and functional changes can be followed in the same animal on long period. In the field of cancer, the dorsal window chamber model is the model of choice for tumor events such as cells migration, vessels growth, and their permeability or interactions between cells and vessels. Coupled with wide-field, confocal, or multiphoton fluorescence microscopes, high spatial and temporal resolutions of the cellular events can be analyzed in vivo.

International expert recommendations on image acquisition for in vivo reflectance confocal microscopy of cutaneous tumors.

BACKGROUND: No international recommendations exist for a minimum imaging requirement per lesion using reflectance confocal microscopy (RCM). This may be beneficial given the increasing use of remote RCM interpretation internationally. OBJECTIVE: To develop international expert recommendations for image acquisition using tissue-coupled RCM for diagnosis of cutaneous tumors. METHODS: Using a modified Delphi approach, a core group developed the scope and drafted initial recommendations before circulation to a larger group, the Cutaneous Imaging Expert Resource Group of the American Academy of Dermatology. Each review round consisted of a period of open comment, followed by revisions. RESULTS: The recommendations were developed after 5 alternating rounds of review among the core group and the Cutaneous Imaging Expert Resource Group. These were divided into subsections of imaging personnel, recommended lesion criteria, clinical and lesion information to be provided, lesion preparation, image acquisition, mosaic cube settings, and additional captures based on lesion characteristics and suspected diagnosis. LIMITATIONS: The current recommendations are limited to tissue-coupled RCM for diagnosis of cutaneous tumors. It is one component of the larger picture of quality assurance and will require ongoing review. CONCLUSIONS: These recommendations serve as a resource to facilitate quality assurance, economical use of time, accurate diagnosis, and international collaboration.

Intravital microscopy visualizes innate immune crosstalk and function in tissue microenvironment.

Significant advances have been made in the field of intravital microscopy (IVM) on myeloid cells due to the growing number of validated fluorescent probes and reporter mice. IVM provides a visualization platform to directly observe cell behavior and deepen our understanding of cellular dynamics, heterogeneity, plasticity, and cell-cell communication in native tissue environments. This review outlines the current studies on the dynamic interaction and function of innate immune cells with a focus on those that are studied with IVM and covers the advances in data analysis with emerging artificial intelligence-based algorithms. Finally, the prospects of IVM on innate immune cells are discussed.

Intravital Microscopy to Study the Effect of Matrix Metalloproteinase Inhibition on Acute Myeloid Leukemia Cell Migration in the Bone Marrow.

Hematopoiesis is the process through which all mature blood cells are formed and takes place in the bone marrow (BM). Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. AML progression causes drastic remodeling of the BM microenvironment, making it no longer supportive of healthy hematopoiesis and leading to clinical cytopenia in patients. Understanding the mechanisms by which AML cells shape the BM to their benefit would lead to the development of new therapeutic strategies. While the role of extracellular matrix (ECM) in solid cancer has been extensively studied during decades, its role in the BM and in leukemia progression has only begun to be acknowledged. In this context, intravital microscopy (IVM) gives the unique insight of direct in vivo observation of AML cell behavior in their environment during disease progression and/or upon drug treatments. Here we describe our protocol for visualizing and analyzing MLL-AF9 AML cell dynamics upon systemic inhibition of matrix metalloproteinases (MMP), combining confocal and two-photon microscopy and focusing on cell migration.

Elucidating Immune Monitoring of Tissue-Resident Macrophages by Intravital Microscopy.

Intravital microscopy is an invaluable tool to study in real time the dynamic behavior of leukocytes in vivo. We describe herein a simple protocol for time-lapse imaging of tissue-resident macrophages in intact kidney, liver, and spleen in live mice. This method can be used in any commercially available inverted confocal microscope, doesn't require expensive lasers or optics, exhibits minimal organ perturbation, photo bleaching, or phototoxicity, and, hence, it enables the study of tissue-resident macrophages in situ and in vivo under steady state and inflammation.

Synchronous Intravital Imaging and Cavitation Monitoring of Antivascular Focused Ultrasound in Tumor Microvasculature Using Monodisperse Low Boiling Point Nanodroplets.

Focused ultrasound-stimulated microbubbles can induce blood flow shutdown and ischemic necrosis at higher pressures in an approach termed antivascular ultrasound. Combined with conventional therapies of chemotherapy, immunotherapy, and radiation therapy, this approach has demonstrated tumor growth inhibition and profound synergistic antitumor effects. However, the lower cavitation threshold of microbubbles can potentially yield off-target damage that the polydispersity of clinical agent may further exacerbate. Here we investigate the use of a monodisperse nanodroplet formulation for achieving antivascular effects in tumors. We first develop stable low boiling point monodisperse lipid nanodroplets and examine them as an alternative agent to mediate antivascular ultrasound. With synchronous intravital imaging and ultrasound monitoring of focused ultrasound-stimulated nanodroplets in tumor microvasculature, we show that nanodroplets can trigger blood flow shutdown and do so with a sharper pressure threshold and with fewer additional events than conventionally used microbubbles. We further leverage the smaller size and prolonged pharmacokinetic profile of nanodroplets to allow for potential passive accumulation in tumor tissue prior to antivascular ultrasound, which may be a means by which to promote selective tumor targeting. We find that vascular shutdown is accompanied by inertial cavitation and complex-order sub- and ultraharmonic acoustic signatures, presenting an opportunity for effective feedback control of antivascular ultrasound.

Transluminal Pillars-Their Origin and Role in the Remodelling of the Zebrafish Caudal Vein Plexus.

Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been described in developing vasculature of many organs and organisms. The aim of this study was to resolve the question about pillar formation and their further maturation employing zebrafish caudal vein plexus (CVP). The CVP development was monitored by in vivo confocal microscopy in high spatio-temporal resolution using the transgenic zebrafish model Fli1a:eGPF//Gata1:dsRed. We tracked back the formation of pillars (diameter ≤ 4 µm) and intercapillary meshes (diameter > 4 µm) and analysed their morphology and behaviour. Transluminal pillars in the CVP arose via a combination of sprouting, lumen expansion, and/or the creation of intraluminal folds, and those mechanisms were not associated directly with blood flow. The follow-up of pillars indicated that one-third of them disappeared between 28 and 48 h post fertilisation (hpf), and of the remaining ones, only 1/17 changed their cross-section area by >50%. The majority of the bigger meshes (39/62) increased their cross-section area by >50%. Plexus simplification and the establishment of hierarchy were dominated by the dynamics of intercapillary meshes, which formed mainly via sprouting angiogenesis. These meshes were observed to grow, reshape, and merge with each other. Our observations suggested an alternative view on intussusceptive angiogenesis in the CVP.

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.