The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Roberto Weigert - One of the best experts on this subject based on the ideXlab platform.
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Nanoarchitecture and dynamics of the mouse enteric glycocalyx examined by freeze-etching electron tomography and Intravital Microscopy
Communications Biology, 2020Co-Authors: Evan S. Krystofiak, Roberto Weigert, Alejandra Leo-macias, Antonio Sesso, Seham Ebrahim, Bechara KacharAbstract:Sun, Krystofiak et al. show the nanoarchitecture of the murine enteric glycocalyx, glycoprotein-rich coat covering cells and assess its porosity and deformability in mice, providing a comprehensive structural framework. This study suggests that the glycocalyx may function as a deformable size-exclusion filter of luminal contents. The glycocalyx is a highly hydrated, glycoprotein-rich coat shrouding many eukaryotic and prokaryotic cells. The intestinal epithelial glycocalyx, comprising glycosylated transmembrane mucins, is part of the primary host-microbe interface and is essential for nutrient absorption. Its disruption has been implicated in numerous gastrointestinal diseases. Yet, due to challenges in preserving and visualizing its native organization, glycocalyx structure-function relationships remain unclear. Here, we characterize the nanoarchitecture of the murine enteric glycocalyx using freeze-etching and electron tomography. Micrometer-long mucin filaments emerge from microvillar-tips and, through zigzagged lateral interactions form a three-dimensional columnar network with a 30 nm mesh. Filament-termini converge into globular structures ~30 nm apart that are liquid-crystalline packed within a single plane. Finally, we assess glycocalyx deformability and porosity using Intravital Microscopy. We argue that the columnar network architecture and the liquid-crystalline packing of the filament termini allow the glycocalyx to function as a deformable size-exclusion filter of luminal contents.
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nanoarchitecture and dynamics of the mouse enteric glycocalyx examined by freeze etching electron tomography and Intravital Microscopy
Communications biology, 2020Co-Authors: Roberto Weigert, Evan S. Krystofiak, Antonio Sesso, Seham Ebrahim, Willy W Sun, Alejandra Leomacias, Runjia Cui, Bechara KacharAbstract:The glycocalyx is a highly hydrated, glycoprotein-rich coat shrouding many eukaryotic and prokaryotic cells. The intestinal epithelial glycocalyx, comprising glycosylated transmembrane mucins, is part of the primary host-microbe interface and is essential for nutrient absorption. Its disruption has been implicated in numerous gastrointestinal diseases. Yet, due to challenges in preserving and visualizing its native organization, glycocalyx structure-function relationships remain unclear. Here, we characterize the nanoarchitecture of the murine enteric glycocalyx using freeze-etching and electron tomography. Micrometer-long mucin filaments emerge from microvillar-tips and, through zigzagged lateral interactions form a three-dimensional columnar network with a 30 nm mesh. Filament-termini converge into globular structures ~30 nm apart that are liquid-crystalline packed within a single plane. Finally, we assess glycocalyx deformability and porosity using Intravital Microscopy. We argue that the columnar network architecture and the liquid-crystalline packing of the filament termini allow the glycocalyx to function as a deformable size-exclusion filter of luminal contents.
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Intravital Microscopy in mammalian multicellular organisms
Current Opinion in Cell Biology, 2019Co-Authors: Seham Ebrahim, Roberto WeigertAbstract:Imaging subcellular processes in live animals is no longer a dream. The development of Intravital Subcellular Microscopy (ISMic) combined with the astounding repertoire of available mouse models makes it possible to investigate processes such as membrane trafficking in mammalian living tissues under native conditions. This has provided the unique opportunity to answer questions that cannot be otherwise addressed in reductionist model systems and to link cell biology to tissue pathophysiology.
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Intravital Microscopy to image membrane trafficking in live rats
Methods of Molecular Biology, 2013Co-Authors: Monika Sramkova, Andrius Masedunskas, Laura Parente, Roberto WeigertAbstract:Intravital Microscopy is a powerful tool that enables imaging various biological processes in live animals. Here, we describe a series of procedures designed to image subcellular structures, such as endosomes and secretory vesicles in the salivary glands (SGs) of live rats. To this aim, we used fluorescently labeled molecules and/or fluorescently tagged proteins that were transiently transfected in the live animal.
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Intravital Microscopy a practical guide on imaging intracellular structures in live animals
Bioarchitecture, 2012Co-Authors: Andrius Masedunskas, Panomwat Amornphimoltham, Oleg Milberg, Natalie Poratshliom, Monika Sramkova, Tim Wigand, Roberto WeigertAbstract:Intravital Microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of Intravital Microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to Intravital Microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cy...
Siqing Shan - One of the best experts on this subject based on the ideXlab platform.
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automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using Intravital Microscopy
American Journal of Physiology-lung Cellular and Molecular Physiology, 2013Co-Authors: Gabi Hanna, Siqing Shan, Andrew N Fontanella, Gregory M Palmer, Daniel R Radiloff, Yulin Zhao, David Irwin, Karyn L Hamilton, Alina Boico, Claude A PiantadosiAbstract:Intravital Microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption Microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the β1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary Intravital Microscopy in laboratory rats for pharmacological and physiological research.
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automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using Intravital Microscopy
American Journal of Physiology-lung Cellular and Molecular Physiology, 2013Co-Authors: Gabi Hanna, Siqing Shan, Andrew N Fontanella, Gregory M Palmer, Daniel R Radiloff, Yulin Zhao, David Irwin, Karyn L Hamilton, Alina Boico, Claude A PiantadosiAbstract:Intravital Microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow....
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mechanisms associated with tumor vascular shut down induced by combretastatin a 4 phosphate Intravital Microscopy and measurement of vascular permeability
Cancer Research, 2001Co-Authors: Gillian M. Tozer, Paul R Barber, Vivien E Prise, John T Wilson, Maja Cemazar, Siqing Shan, Mark W Dewhirst, Borivoj Vojnovic, David J ChaplinAbstract:The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using Intravital Microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using Nω-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to<5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to ∼160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.
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mechanisms associated with tumor vascular shut down induced by combretastatin a 4 phosphate Intravital Microscopy and measurement of vascular permeability
Cancer Research, 2001Co-Authors: Gillian M. Tozer, Paul R Barber, Vivien E Prise, John T Wilson, Maja Cemazar, Siqing Shan, Mark W Dewhirst, Borivoj Vojnovic, David J ChaplinAbstract:The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using Intravital Microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.
David J Chaplin - One of the best experts on this subject based on the ideXlab platform.
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mechanisms associated with tumor vascular shut down induced by combretastatin a 4 phosphate Intravital Microscopy and measurement of vascular permeability
Cancer Research, 2001Co-Authors: Gillian M. Tozer, Paul R Barber, Vivien E Prise, John T Wilson, Maja Cemazar, Siqing Shan, Mark W Dewhirst, Borivoj Vojnovic, David J ChaplinAbstract:The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using Intravital Microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using Nω-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to<5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to ∼160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.
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mechanisms associated with tumor vascular shut down induced by combretastatin a 4 phosphate Intravital Microscopy and measurement of vascular permeability
Cancer Research, 2001Co-Authors: Gillian M. Tozer, Paul R Barber, Vivien E Prise, John T Wilson, Maja Cemazar, Siqing Shan, Mark W Dewhirst, Borivoj Vojnovic, David J ChaplinAbstract:The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using Intravital Microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.
Laila Ritsma - One of the best experts on this subject based on the ideXlab platform.
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in vivo imaging and histochemistry are combined in the cryosection labelling and Intravital Microscopy technique
Nature Communications, 2013Co-Authors: Laila Ritsma, Nienke Vrisekoop, Jacco Van RheenenAbstract:Imaging cells within live animals is important in biomedical research, but this process is limited by the availability of probes. Ritsma et al. combine Intravital Microscopy with immunohistochemistry to increase the panel of available reagents and examine the role of T cells in the migration of breast cancer cells.
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Surgical implantation of an abdominal imaging window for Intravital Microscopy
Nature Protocols, 2013Co-Authors: Laila Ritsma, Ernst J A Steller, Saskia I J Ellenbroek, Onno Kranenburg, Inne H M Borel Rinkes, Jacco Van RheenenAbstract:High-resolution Intravital Microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; however, long-term imaging studies on cellular processes in abdominal organs are more challenging. These processes include colonization of the liver by metastatic tumor cells and the development of an immune response in the spleen. We have recently developed an abdominal imaging window (AIW) that allows long-term imaging of the liver, the pancreas, the intestine, the kidney and the spleen. Here we describe the detailed protocol for the optimal surgical implantation of the AIW, which takes ∼1 h, and subsequent multiphoton imaging, which takes up to 1 month.
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Intravital Microscopy through an abdominal imaging window reveals a pre micrometastasis stage during liver metastasis
Science Translational Medicine, 2012Co-Authors: Laila Ritsma, Nienke Vrisekoop, Evelyne Beerling, Ernst J A Steller, Cindy J M Loomans, Anoek Zomer, Carmen Gerlach, Danielle Seinstra, Leon Van Gurp, Ronny SchaferAbstract:Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with Intravital Microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.
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Intravital Microscopy new insights into metastasis of tumors
Journal of Cell Science, 2011Co-Authors: Evelyne Beerling, Laila Ritsma, Nienke Vrisekoop, Patrick W B Derksen, Jacco Van RheenenAbstract:Metastasis, the process by which cells spread from the primary tumor to a distant site to form secondary tumors, is still not fully understood. Although histological techniques have provided important information, they give only a static image and thus compromise interpretation of this dynamic process. New advances in Intravital Microscopy (IVM), such as two-photon Microscopy, imaging chambers, and multicolor and fluorescent resonance energy transfer imaging, have recently been used to visualize the behavior of single metastasizing cells at subcellular resolution over several days, yielding new and unexpected insights into this process. For example, IVM studies showed that tumor cells can switch between multiple invasion strategies in response to various densities of extracellular matrix. Moreover, other IVM studies showed that tumor cell migration and blood entry take place not only at the invasive front, but also within the tumor mass at tumor-associated vessels that lack an intact basement membrane. In this Commentary, we will give an overview of the recent advances in high-resolution IVM techniques and discuss some of the latest insights in the metastasis field obtained with IVM.
Ralph Weissleder - One of the best experts on this subject based on the ideXlab platform.
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single cell Intravital Microscopy of trastuzumab quantifies heterogeneous in vivo kinetics
Cytometry Part A, 2019Co-Authors: Adel Attari, Ralph Weissleder, Mark Prytyskach, Michelle A Garlin, Miles A MillerAbstract:Cell-to-cell heterogeneity can substantially impact drug response, especially for monoclonal antibody (mAb) therapies that may exhibit variability in both delivery (pharmacokinetics) and action (pharmacodynamics) within solid tumors. However, it has traditionally been difficult to examine the kinetics of mAb delivery at a single-cell level and in a manner that enables controlled dissection of target-dependent and -independent behaviors. To address this issue, here we developed an in vivo confocal (Intravital) Microscopy approach to study single-cell mAb pharmacology in a mosaic xenograft comprising a mixture of cancer cells with variable expression of the receptor HER2. As a proof-of-principle, we applied this model to trastuzumab therapy, a HER2-targeted mAb widely used for treating breast and gastric cancer patients. Trastuzumab accumulated to a higher degree in HER2-over expressing tumor cells compared to HER2-low tumor cells (~5:1 ratio at 24 h after administration) but importantly, the majority actually accumulated in tumor-associated phagocytes. For example, 24 h after IV administration over 50% of tumoral trastuzumab was found in phagocytes whereas at 48 h it was >80%. Altogether, these results reveal the dynamics of how phagocytes influence mAb behavior in vivo, and demonstrate an application of Intravital Microscopy for quantitative single-cell measurement of mAb distribution and retention in tumors with heterogeneous target expression. © 2019 International Society for Advancement of Cytometry.
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imaging the pharmacology of nanomaterials by Intravital Microscopy toward understanding their biological behavior
Advanced Drug Delivery Reviews, 2017Co-Authors: Miles A Miller, Ralph WeisslederAbstract:Therapeutic nanoparticles (NPs) can deliver cytotoxic chemotherapeutics and other drugs more safely and efficiently to patients; furthermore, selective delivery to target tissues can theoretically be accomplished actively through coating NPs with molecular ligands, and passively through exploiting physiological "enhanced permeability and retention" features. However, clinical trial results have been mixed in showing improved efficacy with drug nanoencapsulation, largely due to heterogeneous NP accumulation at target sites across patients. Thus, a clear need exists to better understand why many NP strategies fail in vivo and not result in significantly improved tumor uptake or therapeutic response. Multicolor in vivo confocal fluorescence imaging (Intravital Microscopy; IVM) enables integrated pharmacokinetic and pharmacodynamic (PK/PD) measurement at the single-cell level, and has helped answer key questions regarding the biological mechanisms of in vivo NP behavior. This review summarizes progress to date and also describes useful technical strategies for successful IVM experimentation.
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motion characterization scheme to minimize motion artifacts in Intravital Microscopy
Journal of Biomedical Optics, 2017Co-Authors: Sungon Lee, Ralph Weissleder, Gabriel Courties, Matthias Nahrendorf, Claudio VinegoniAbstract:Respiratory- and cardiac-induced motion artifacts pose a major challenge for in vivo optical imaging, limiting the temporal and spatial imaging resolution in fluorescence laser scanning Microscopy. Here, we present an imaging platform developed for in vivo characterization of physiologically induced axial motion. The motion characterization system can be straightforwardly implemented on any conventional laser scanning microscope and can be used to evaluate the effectiveness of different motion stabilization schemes. This method is particularly useful to improve the design of novel tissue stabilizers and to facilitate stabilizer positioning in real time, therefore facilitating optimal tissue immobilization and minimizing motion induced artifacts.
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imaging the beating heart in the mouse using Intravital Microscopy techniques
Nature Protocols, 2015Co-Authors: Claudio Vinegoni, Aaron D Aguirre, Sungon Lee, Ralph WeisslederAbstract:Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits Intravital Microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for Intravital Microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution Microscopy studies.
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automated motion artifact removal for Intravital Microscopy without a priori information
Scientific Reports, 2015Co-Authors: Sungon Lee, Claudio Vinegoni, Matthew Sebas, Ralph WeisslederAbstract:Intravital fluorescence Microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during Intravital Microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton Microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.
