Intravital Imaging Research Articles

Plasmodium sporozoite search strategy to locate hotspots of blood vessel invasion

Plasmodium sporozoites actively migrate in the dermis and enter blood vessels to infect the liver. Despite their importance for malaria infection, little is known about these cutaneous processes. We combine intravital imaging in a rodent malaria model and statistical methods to unveil the parasite strategy to reach the bloodstream. We determine that sporozoites display a high-motility mode with a superdiffusive Lévy-like pattern known to optimize the location of scarce targets. When encountering blood vessels, sporozoites frequently switch to a subdiffusive low-motility behavior associated with probing for intravasation hotspots, marked by the presence of pericytes. Hence, sporozoites present anomalous diffusive motility, alternating between superdiffusive tissue exploration and subdiffusive local vessel exploitation, thus optimizing the sequential tasks of seeking blood vessels and pericyte-associated sites of privileged intravasation.

Nanoparticle-based targeting of microglia improves the neural regeneration enhancing effects of immunosuppression in the zebrafish retina

Retinal Müller glia function as injury-induced stem-like cells in zebrafish but not mammals. However, insights gleaned from zebrafish have been applied to stimulate nascent regenerative responses in the mammalian retina. For instance, microglia/macrophages regulate Müller glia stem cell activity in the chick, zebrafish, and mouse. We previously showed that post-injury immunosuppression by the glucocorticoid dexamethasone accelerated retinal regeneration kinetics in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation of microglia reactivity may therefore enhance the regenerative potential of Müller glia for therapeutic purposes. Here, we investigated potential mechanisms by which post-injury dexamethasone accelerates retinal regeneration kinetics, and the effects of dendrimer-based targeting of dexamethasone to reactive microglia. Intravital time-lapse imaging revealed that post-injury dexamethasone inhibited microglia reactivity. The dendrimer-conjugated formulation: (1) decreased dexamethasone-associated systemic toxicity, (2) targeted dexamethasone to reactive microglia, and (3) improved the regeneration enhancing effects of immunosuppression by increasing stem/progenitor proliferation rates. Lastly, we show that the gene rnf2 is required for the enhanced regeneration effect of D-Dex. These data support the use of dendrimer-based targeting of reactive immune cells to reduce toxicity and enhance the regeneration promoting effects of immunosuppressants in the retina.

LPS-Induced Systemic Inflammation Affects the Dynamic Interactions of Astrocytes and Microglia with the Vasculature of the Mouse Brain Cortex.

The Neurovascular Unit (NVU), composed of glia (astrocytes, oligodendrocytes, microglia), neurons, pericytes and endothelial cells, is a dynamic interface ensuring the physiological functioning of the central nervous system (CNS), which gets affected and contributes to the pathology of several neurodegenerative diseases. Neuroinflammation is a common feature of neurodegenerative diseases and is primarily related to the activation state of perivascular microglia and astrocytes, which constitute two of its major cellular components. Our studies focus on monitoring in real time the morphological changes of perivascular astrocytes and microglia, as well as their dynamic interactions with the brain vasculature, under physiological conditions and following systemic neuroinflammation triggering both microgliosis and astrogliosis. To this end, we performed 2-photon laser scanning microscopy (2P-LSM) for intravital imaging of the cortex of transgenic mice visualizing the dynamics of microglia and astroglia following neuroinflammation induced by systemic administration of the endotoxin lipopolysaccharide (LPS). Our results indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, an event that most possibly contributes to a loss of blood-brain barrier (BBB) integrity. At the same time, microglial cells become activated and exhibit a higher extent of physical contact with the blood vessels. These dynamic responses of perivascular astrocytes and microglia are peaking at 4 days following LPS administration; however, they still persist at a lower level at 8 days after LPS injection, revealing incomplete reversal of inflammation affecting the glial properties and interactions within the NVU.

Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice.

Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.

Intravital Longitudinal Three-Photon Imaging of Lymphocyte Migration in the Mouse Spleen.

Abstract For the last 20 years, intravital two-photon (2P) imaging has been a powerful tool to visualize lymphocyte dynamics in mouse lymphoid organs such as bone marrows and lymph nodes. Although spleen is a key lymphoid organ for adaptive immunity against blood-derived antigens, the lymphocyte dynamics in the spleen are poorly understood because intravital 2P imaging of spleen is challenging. Visualizing the white pulp in the spleen where most lymphocytes are located requires the excitation light to propagate through the red pulp which contains a lot of blood, causing high light-scattering. In addition, intravital spleen imaging suffers from motion artifact due to the mouse breathing motion. To overcome these challenges, we applied three-photon (3P) microscopy for spleen imaging, which can reduce light scattering and improve image contrast in deeper regions compared to 2P microscopy. We also designed a new implantable, chronic imaging window for reducing spleen motion in our up-light microscope. We could identify white pulp regions by in vivo labeling of the marginal zone of white pulp. Using 3P microscopy at 1300 nm wavelength, we could visualize EGFP+ lymphocytes in white pulps at approximately 400–500 μm depth below the surface while 2P microscopy at 920 nm could only reach imaging depths of 160–250 μm in the same region. We then determined the temperature of the imaging window that is required for maintaining normal lymphocyte motility during imaging by monitoring the lymphocyte motility at different temperatures. This intravital longitudinal 3P imaging technique has the potential to examine lymphocyte dynamics in blood-derived antigen-induced inflammatory process in the spleen.

In vivo imaging system for cellular level visualization of immune responses in various animal models

Abstract Immune cells have a variety of functions to defend against pathogens under certain circumstances. It is not until recently, However, that these miscellaneous responses can be observed in vivo. Herein, we optimized some solutions of researchers keeping track of the changes of the immune response by utilizing the IVM-C intravital imaging system and in vivo fluorescence labeling for target cells. It is possible to successfully acquire a crystal-clear cellular level images of immune cells in the different animal model including RNA adjuvant-injected mouse and sepsis-induced one by using intravital microscope, respectively. In case of the RNA adjuvant injection, it is obvious that retention of the antigen presenting cells in draining lymph node is on the rise to promote T/B cell activation. A dramatic increase in the number of the infiltrated dendritic cells into the organ was observed at the same time. Moreover, Pathogenesis of sepsis by monitoring of pulmonary capillary circulation was investigated. In order to visualize pulmonary microcirculation, we’ve established a pulmonary imaging window which makes it feasible to image the real-time dynamics of rapidly circulating neutrophils in live mouse. We found out that many clusters of neutrophils aggregates in the pulmonary vasculature or, even in the arteriole of the sepsis-induced mouse, thereby disturbing the microcirculation of that region and generating dead space in the pulmonary structure. Taking everything into account, we make certain it is appropriate to utilize the intravital imaging method for cellular level visualization of the immune system under various pathological conditions. we believe that it could be an invaluable tool for a comprehensive understanding of immunology.

Dynamics of lymphoid-mammary epithelial cell interaction during pregnancy and lactation

Abstract Breast milk has been shown to play a key role in the transfer of immunity from mother to offspring. Antibodies and lymphoid cells can be passed through breast milk and impact offspring immunity. However, the role of these lymphoid cells in lactation and in the control of offspring immunity remains unclear. We observed that the lymphoid cells present in the breast milk are distinct from those found in the blood. Compared to the blood, breast milk T cells are enriched in a T-bet +unconventional population. Imaging analysis revealed that these T-bet +cells gradually accumulate in the mammary gland during pregnancy and are specifically localized around and inside the mammary epithelium. We will utilize confocal and intravital imaging of the mammary gland in virgin, pregnant, and lactating mice to characterize the dynamics of this mammary-associated T-bet +population throughout pregnancy and lactation. Understanding the dynamics of T-bet +cells and their anatomical localization within the mammary gland will help us decode how these cells impact the remodeling of the mammary epithelium during pregnancy and lactation. We propose that the remodeling of the mammary gland imparted by the immune system can also influence the transfer of immunity from mother to offspring, thereby affecting the development of offspring immunity. This work was supported in part by intramural funds of NIAID, NIH.

Dual TLR9 and PD-L1 targeting unleashes dendritic cells to induce durable antitumor immunity

BackgroundAlthough immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may...

Sympathetic regulation of skin microbiota-induced innate-like T cells

Abstract Recent discoveries in microbiota-mediated neuroimmune crosstalk highlight how co-evolved systems collaboratively facilitate host fitness. However, in the skin – the largest and outermost protective barrier – little is known about the role of microbiota in neuro-immune regulation. We find that murine skin colonization with commensal Staphylococci induces significant accumulation of dermal IL-17A-producing Vg6 +gdT (Vg6 gdT17) cells in IL-1 and microbiota-dependent manner. Compared to sedentary Vg6 gdT17 from unassociated controls, intravital imaging shows that commensal-induced dermal Vg6 gdT17 form large highly motile aggregates. Transcriptomic analysis revealed significant differences between Vg6 gdT17 from commensal-colonized mice versus unassociated controls. Among these, the cells from colonized mice showed significant downregulation of sympathetic beta-adrenoreceptor (bAR) expression. Beta-AR signaling has recently been shown to impact conventional T cell motility in the skin. We observe that, upon systemic b2AR agonist administration, the highly dynamic commensal-induced dermal Vg6 +gdT17 clusters are rendered immobile, showing sympathetic control of gdT17 cell motility. Intriguingly, skin commensal exposure leads to systemic induction of Vg6 +gdT17 cells, implying a role for these cells beyond local surveillance. Further studies are underway to test whether, following exposure to skin commensal, sympathetic regulation promotes inter-organ gdT cell dissemination and impacts their functional potential. Overall, this work aims to uncover the mechanism(s) underlying microbiota-induced skin neuroimmune interactions in promoting organismal homeostasis.

Plasmodium-infected mosquito bite elicits an immune response at the bite site

Abstract To induce long-lasting immunity, the host’s immune system must recognize and respond to an invading pathogen upon first encounter. Plasmodium sporozoites, the infective stage of the malaria parasite, are inoculated into the skin by infected mosquitoes as they probe for blood. Only 20% of sporozoites reach the liver, while the rest either reach the draining lymph nodes (dLN) or remain in the infected area for at least 6 hours, shedding an abundant amount of their major surface protein, the circumsporozoite protein (CSP). Yet, sterile immunity against sporozoites or CSP does not occur, despite the presence of the foreign antigens in the skin and the dLN, and repeated exposures, suggesting that Plasmodium sporozoites may modulate the host immune response. In this study we investigate the immune response elicited by P. berghei-infected mosquito bite at the bite site in-vivo. Using intravital imaging of sporozoites inoculation by a mosquito bite to a LysM-CRE GFP expressing mouse, we can observe neutrophil recruitment to the bite site in real time. When investigating the expression of pro- and anti-inflammatory genes in the skin, we find a specific expression patten that is elevated in response to a P. berghei-infected mosquito bite, when compared to a bite of an uninfected mosquito. Here we show fast immune cells recruitment as well as specific immune response to Plasmodium infection, suggesting a quick recognition of infection by the immune system. Nonetheless, gene expression analysis suggests a mechanism by which Plasmodium sporozoites may suppress the immune response to their presence. Kindly supported by grants from NIH (R01 AI132359) and JHMRI.

Real-Time Intravital Imaging of Acoustic Cluster Therapy-Induced Vascular Effects in the Murine Brain.

The blood-brain barrier (BBB) is an obstacle for cerebral drug delivery. Controlled permeabilization of the barrier by external stimuli can facilitate the delivery of drugs to the brain. Acoustic Cluster Therapy (ACT®) is a promising strategy for transiently and locally increasing the permeability of the BBB to macromolecules and nanoparticles. However, the mechanism underlying the induced permeability change and subsequent enhanced accumulation of co-injected molecules requires further elucidation. In this study, the behavior of ACT® bubbles in microcapillaries in the murine brain was observed using real-time intravital multiphoton microscopy. For this purpose, cranial windows aligned with a ring transducer centered around an objective were mounted to the skull of mice. Dextrans labeled with 2 MDa fluorescein isothiocyanate (FITC) were injected to delineate the blood vessels and to visualize extravasation. Activated ACT® bubbles were observed to alter the blood flow, inducing transient and local increases in the fluorescence intensity of 2 MDa FITC-dextran and subsequent extravasation in the form of vascular outpouchings. The observations indicate that ACT® induced a transient vascular leakage without causing substantial damage to the vessels in the brain. The study gave novel insights into the mechanism underlying ACT®-induced enhanced BBB permeability which will be important considering treatment optimization for a safe and efficient clinical translation of ACT®.

Two-photon synthetic aperture microscopy for minimally invasive fast 3D imaging of native subcellular behaviors in deep tissue

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.

Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse.

Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.

Magnetic resonance/fluorescence dual-modality contrast agents targeting αvβ6-overexpressing tumors based on A20FMDV2 peptide as a ligand

Pancreatic ductal adenocarcinoma (PDAC) is a malignant digestive system tumor with a poor late-stage prognosis. This study aimed to identify new methods for the early detection of PDAC. The nanoprobe A20FMDV2-Gd-5-FAM was developed using A20FMDV2 (N1AVPNLRGDLQVLAQKVART20-NH2, A20FMDV2) as the ligand and characterized using dynamic light scattering, transmission electron microscopy, Fourier transform infrared analysis, and UV absorption spectroscopy. The binding of pancreatic cancer cells AsPC-1, MIA PaCa-2, and normal human pancreatic H6C7 cells (HPDE6-C7) to the probe was verified using laser confocal microscopy, and the biocompatibility of the probe was evaluated in vivo. In vivo magnetic resonance and fluorescence imaging were also performed on nude mice with subcutaneous pancreatic tumor xenografts to verify the bimodal imaging performance of the probe. The probe exhibited good stability and biocompatibility and an enhanced relaxation rate (25.46 ± 1.32 mM−1 s−1) than Gd-DTPA. Confocal laser scanning microscopy results revealed that the A20FMDV2-Gd-5-FAM probe could be successfully ingested and internalized, and infrared analysis results demonstrated that the probe was linked successfully. Finally, magnetic resonance T1WI imaging and intravital fluorescence imaging demonstrated the specific signal enhancement of the probe at the tumor site. In conclusion, the bimodal molecular probe A20FMDV2-Gd-5-FAM showed a stable magnetic resonance and fluorescence bimodal imaging performance and is a promising new approach for diagnosing early-stage cancers with a high integrin αvβ6 expression.

Intravenously administered APAC, a dual AntiPlatelet AntiCoagulant, targets arterial injury site to inhibit platelet thrombus formation and tissue factor activity in mice

IntroductionArterial thrombosis is the main underlying mechanism of acute atherothrombosis. Combined antiplatelet and anticoagulant regimens prevent thrombosis but increase bleeding rates. Mast cell-derived heparin proteoglycans have local antithrombotic properties, and their semisynthetic dual AntiPlatelet and AntiCoagulant (APAC) mimetic may provide a new efficacious and safe tool for arterial thrombosis. We investigated the in vivo impact of intravenous APAC (0.3–0.5 mg/kg; doses chosen according to pharmacokinetic studies) in two mouse models of arterial thrombosis and the in vitro actions in mouse platelets and plasma. Materials and methodsPlatelet function and coagulation were studied with light transmission aggregometry and clotting times. Carotid arterial thrombosis was induced either by photochemical injury or surgically exposing vascular collagen after infusion of APAC, UFH or vehicle. Time to occlusion, targeting of APAC to the vascular injury site and platelet deposition on these sites were assessed by intra-vital imaging. Tissue factor activity (TF) of the carotid artery and in plasma was captured. ResultsAPAC inhibited platelet responsiveness to agonist stimulation (collagen and ADP) and prolonged APTT and thrombin time. After photochemical carotid injury, APAC-treatment prolonged times to occlusion in comparison with UFH or vehicle, and decreased TF both in carotid lysates and plasma. Upon binding from circulation to vascular collagen-exposing injury sites, APAC reduced the in situ platelet deposition. ConclusionsIntravenous APAC targets arterial injury sites to exert local dual antiplatelet and anticoagulant actions and attenuates thrombosis upon carotid injuries in mice. Systemic APAC provides local efficacy, highlighting APAC as a novel antithrombotic to reduce cardiovascular complications.

Immunomodulation under the lens of real-time in vivo imaging.

Modulation of cells and molecules of the immune system not only represents a major opportunity to treat a variety of diseases including infections, cancer, autoimmune, and inflammatory disorders but could also help understand the intricacies of immune responses. A detailed mechanistic understanding of how a specific immune intervention may provide clinical benefit is essential for the rational design of efficient immunomodulators. Visualizing the impact of immunomodulation in real-time and in vivo has emerged as an important approach to achieve this goal. In this review, we aim to illustrate how multiphoton intravital imaging has helped clarify the mode of action of immunomodulatory strategies such as antibodies or cell therapies. We also discuss how optogenetics combined with imaging will further help manipulate and precisely understand immunomodulatory pathways. Combined with other single-cell technologies, in vivo dynamic imaging has therefore a major potential for guiding preclinical development of immunomodulatory drugs.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

Ultrasonic enhancement of microdroplet-based interfacial reaction for improving the synthesis of Ag2S QDs

Ag2S quantum dots (QDs) have aroused extensive concerns in intravital imaging field due to their merits of narrow bandgap, low biological toxicity and decent fluorescence emission properties in the second near-infrared (NIR-II) window. However, low quantum yield (QY) and poor uniformity of Ag2S QDs are still main obstacles for its application. In this work, a novel strategy of utilizing ultrasonic field is presented, which can enhance the microdroplet-based interfacial synthesis of Ag2S QDs. The ultrasound increases the presence of ions at the reaction sites by enhancing the ion mobility in the microchennels. Therefore, the QY is enhanced from 2.33 % (optimal QY without ultrasound) to 8.46 %, which is the highest value of Ag2S ever reported without ion-doping. Also, the decrease of the corresponding full width at half maximum (FWHM) from 312 nm to 144 nm indicates the obvious uniformity improvement of the obtained QDs. Further mechanism exploration illustrates that ultrasonic cavitation significantly increases the interfacial reaction sites by splitting the droplets. Meanwhile, the acoustic flow field strengthens the ion renewal at the droplet interface. Consequently, the mass transfer coefficient increases by more than 500 %, which is favorable to improve both the QY and quality of Ag2S QDs. This work serves both fundamental research and practical production for the synthesis of Ag2S QDs.

Monitoring the perivascular cerebrospinal fluid dynamics of the glymphatic pathway using co-localized photoacoustic microscopy.

In vivo imaging plays an important role in investigating how the glymphatic system drains metabolic waste and pathological proteins from the central nervous system. However, the spatial resolutions and imaging specificities of the available preclinical imaging methods for the glymphatic system are insufficient, and they cannot simultaneously locate the cerebrovascular and glymphatic pathways to enable the monitoring of the perivascular cerebrospinal fluid dynamics. This Letter proposes an imaging strategy for the in vivo monitoring of cerebrospinal fluid flow using co-localized photoacoustic volumetric microscopy. Imaging results showed that the glymphatic pathway is one of the crucial pathways for the drainage of cerebrospinal fluid, and it mainly enters the brain parenchyma along periarterial routes. Continuous intravital imaging enables the monitoring of the cerebrospinal fluid flow as well as the drainage and clearance from the glymphatic system after the tracer has entered the cerebrospinal fluid. The technique can enhance understanding of the cerebrospinal fluid circulation and open up new insights into neurodegenerative brain diseases.