IU Indianapolis ScholarWorks :: Browsing by Subject "Intravital microscopy"

Browsing by Subject "Intravital microscopy"

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An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue
(BMC, 2013-07-03) O’Connor, Amber K.; Malarkey, Erik B.; Berbari, Nicolas F.; Croyle, Mandy J.; Haycraft, Courtney J.; Bell, P. Darwin; Hohenstein, Peter; Kesterson, Robert A.; Yoder, Bradley K.; Biology, School of Science
Background: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. Results: To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. Conclusions: The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.
Digital Image Analysis Tools Developed by the Indiana O’Brien Center
(Frontiers Media, 2021-12-16) Dunn, Kenneth W.; Medicine, School of Medicine
The scale and complexity of images collected in biological microscopy have grown enormously over the past 30 years. The development and commercialization of multiphoton microscopy has promoted a renaissance of intravital microscopy, providing a window into cell biology in vivo. New methods of optical sectioning and tissue clearing now enable biologists to characterize entire organs at subcellular resolution. New methods of multiplexed imaging support simultaneous localization of forty or more probes at a time. Exploiting these exciting new techniques has increasingly required biomedical researchers to master procedures of image analysis that were once the specialized province of imaging experts. A primary goal of the Indiana O'Brien Center has been to develop robust and accessible image analysis tools for biomedical researchers. Here we describe biomedical image analysis software developed by the Indiana O'Brien Center over the past 25 years.
Distinct behavior of myelomonocytic cells and CD8 T cells underlies the hepatic response to Listeria monocytogenes
(F1000Research, 2018-04-24) Velázquez, Peter; Williams, Cassandra; Leiner, Ingrid; Pamer, Eric G.; Dustin, Michael L.; Microbiology and Immunology, School of Medicine
Background: The immune response to Listeria monocytogenes (LM) is characterized by formation of leukocyte rich foci of infection in liver and spleen. Although much has been gained in our understanding of immune response through the study of LM, little is known about spatio-temporal regulation of immune response to Listeria in liver. Methods: We utilize a combination of molecular, genetic and intravital microscopic approaches to gain insight into the dynamics of foci and leukocyte behavior during hepatic Listeriosis. Results: LM foci efficiently exclude blood flow, indicating the presence of a barrier separating the foci and healthy tissue. Despite this barrier, sinusoidal myelomonocytic cells readily enter or transiently interact with cells at the edge of foci of infection. Next, utilizing L9.6 transgenic CD8 + T cells specific for an endogenously processed LM antigen, p60 217-225, along with LM deficient in this epitope, we define the role of TCR in T cell migratory behavior in infected liver. Surprisingly, T cell behavior varies with micro-anatomic locale. Near foci, non-specific adhesion mechanisms dominate lymphocyte behavior. Antigen specific effects on motility became detectable only distal to foci. Conclusions: These data suggest that LM antigens act in a paracrine manner to mediate protection from Listeriosis in the liver.
The Indiana O'Brien Center for Advanced Renal Microscopic Analysis
(American Physiological Society, 2021) Dunn, Kenneth W.; Molitoris, Bruce A.; Dagher, Pierre C.; Medicine, School of Medicine
The Indiana O'Brien Center for Advanced Microscopic Analysis is a National Institutes of Health (NIH) P30-funded research center dedicated to the development and dissemination of advanced methods of optical microscopy to support renal researchers throughout the world. The Indiana O'Brien Center was founded in 2002 as an NIH P-50 project with the original goal of helping researchers realize the potential of intravital multiphoton microscopy as a tool for understanding renal physiology and pathophysiology. The center has since expanded into the development and implementation of large-scale, high-content tissue cytometry. The advanced imaging capabilities of the center are made available to renal researchers worldwide via collaborations and a unique fellowship program. Center outreach is accomplished through an enrichment core that oversees a seminar series, an informational website, and a biennial workshop featuring hands-on training from members of the Indiana O'Brien Center and imaging experts from around the world.
Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance
(American Physiological Society, 2021) Yadav, Shiv Pratap S.; Sandoval, Ruben M.; Zhao, Jingfu; Huang, Yifan; Wang, Exing; Kumar, Sudhanshu; Campos-Bilderback, Silvia B.; Rhodes, George; Mechref, Yehia; Molitoris, Bruce A.; Wagner, Mark C.; Medicine, School of Medicine
Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.
Mitochondrial depolarization and repolarization in the early stages of acetaminophen hepatotoxicity in mice
(Elsevier, 2020-06) Dunn, Kenneth W.; Martinez, Michelle M.; Wang, Zemin; Mang, Henry E.; Clendenon, Sherry G.; Sluka, James P.; Glazier, James A.; Klaunig, James E.; Medicine, School of Medicine
Mitochondrial injury and depolarization are primary events in acetaminophen hepatotoxicity. Previous studies have shown that restoration of mitochondrial function in surviving hepatocytes, which is critical to recovery, is at least partially accomplished via biogenesis of new mitochondria. However, other studies indicate that mitochondria also have the potential to spontaneously repolarize. Although repolarization was previously observed only at a sub-hepatotoxic dose of acetaminophen, we postulated that mitochondrial repolarization in hepatocytes outside the centrilobular regions of necrosis might contribute to recovery of mitochondrial function following acetaminophen-induced injury. Our studies utilized longitudinal intravital microscopy of millimeter-scale regions of the mouse liver to characterize the spatio-temporal relationship between mitochondrial polarization and necrosis early in acetaminophen-induced liver injury. Treatment of male C57BL/6J mice with a single intraperitoneal 250 mg/kg dose of acetaminophen resulted in hepatotoxicity that was apparent histologically within 2 h of treatment, leading to 20 and 60-fold increases in serum aspartate aminotransferase and alanine aminotransferase, respectively, within 6 h. Intravital microscopy of the livers of mice injected with rhodamine123, TexasRed-dextran, propidium iodide and Hoechst 33342 detected centrilobular foci of necrosis within extended regions of mitochondrial depolarization within 2 h of acetaminophen treatment. Although regions of necrosis were more apparent 6 h after acetaminophen treatment, the vast majority of hepatocytes with depolarized mitochondria did not progress to necrosis, but rather recovered mitochondrial polarization within 6 h. Recovery of mitochondrial function following acetaminophen hepatotoxicity thus involves not only biogenesis of new mitochondria, but also repolarization of existing mitochondria. These studies also revealed a spatial distribution of necrosis and mitochondrial depolarization whose single-cell granularity is inconsistent with the hypothesis that communication between neighboring cells plays an important role in the propagation of necrosis during the early stages of APAP hepatotoxicity. Small islands of healthy, intact cells were frequently found surrounded by necrotic cells, and small islands of necrotic cells were frequently found surrounded by healthy, intact cells. Time-series studies demonstrated that these "islands", consisting in some cases of single cells, are persistent; over a period of hours, injury does not spread from individual necrotic cells to their neighbors.
Mouse models and human islet transplantation sites for intravital imaging
(Frontiers Media, 2022-10-05) Wagner, Leslie E.; Melnyk, Olha; Duffett, Bryce E.; Linnemann, Amelia K.; Biochemistry and Molecular Biology, School of Medicine
Human islet transplantations into rodent models are an essential tool to aid in the development and testing of islet and cellular-based therapies for diabetes prevention and treatment. Through the ability to evaluate human islets in an in vivo setting, these studies allow for experimental approaches to answer questions surrounding normal and disease pathophysiology that cannot be answered using other in vitro and in vivo techniques alone. Intravital microscopy enables imaging of tissues in living organisms with dynamic temporal resolution and can be employed to measure biological processes in transplanted human islets revealing how experimental variables can influence engraftment, and transplant survival and function. A key consideration in experimental design for transplant imaging is the surgical placement site, which is guided by the presence of vasculature to aid in functional engraftment of the islets and promote their survival. Here, we review transplantation sites and mouse models used to study beta cell biology in vivo using intravital microscopy and we highlight fundamental observations made possible using this methodology.
A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy
(American Psychological Society, 2015-12-01) Tao, Wen; Rubart, Michael; Ryan, Jennifer; Xiao, Xiao; Qiao, Chunping; Hato, Takashi; Davidson, Michael W.; Dunn, Kenneth W.; Day, Richard N.; Department of Medicine, IU School of Medicine
The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.
A simple automated method for continuous fieldwise measurement of microvascular hemodynamics
(Elsevier, 2019-05) Clendenon, Sherry G.; Fu, Xiao; Von Hoene, Robert A.; Clendenon, Jeffrey L.; Sluka, James P.; Winfree, Seth; Mang, Henry; Martinez, Michelle; Filson, Adele J.; Klaunig, James E.; Glazier, James A.; Dunn, Kenneth W.; Medicine, School of Medicine
Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using high-speed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.

Browsing by Subject "Intravital microscopy"

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