Browsing by Subject "Confocal microscopy"
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Item Diversity of two forms of DNA methylation in the brain(Frontiers Media, 2014-03-10) Chen, Yuanyuan; Damayanti, Nur P.; Irudayaraj, Joseph; Dunn, Kenneth; Zhou, Feng C.; Anatomy, Cell Biology and Physiology, School of MedicineDNA methylation 5-methylcytosine (5mC) predicts a compacting chromatin inaccessible to transcription. The discovery of 5-hydroxymethylcytosine (5hmC), which is derived from 5mC, adds a new dimension to the mechanism and role of DNA methylation in epigenetics. Genomic evidence indicates that the 5hmC is located in the alternate regions to 5mC. However, the nature of 5hmC, as compared with classical 5mC remains unclear. Observing the mouse brain through embryonic development to the adult, first, we found that 5hmC is not merely an intermediate metabolite of demethylation, but is long lasting, chromatically distinct, and dynamically changing during neurodevelopment. Second, we found that 5hmC distinctly differs from 5mC in its chromatin affiliation during neural stem cell (NSC) development. Thirdly, we found both 5mC and 5hmC to be uniquely polarized and dynamic through the NSC development. 5mC was found to progressively polarize with MBD1 and MeCP2, and recruits H3K9me3 and H3K27me3; while 5hmC progressively co-localizes with MBD3 and recruits H3K4me2. Critical differential binding of 5mC with MBD1, and 5hmC with MBD3 was validated by Resonance Energy Transfer technique FLIM-FRET. This transition and polarization coincides with neuroprogenitor differentiation. Finally, at the time of synaptogenesis, 5mC gradually accumulates in the heterochromatin while 5hmC accumulates in the euchromatin, which is consistent with the co-localization of 5hmC with PolII, which mediates RNA transcription. Our data indicate that 5mC and 5hmC are diverse in their functional interactions with chromatin. This diversity is likely to contribute to the versatile epigenetic control of transcription mediating brain development and functional maintenance of adult brain.Item Geometric Navigation of Axons in a Cerebral Pathway: Comparing dMRI with Tract Tracing and Immunohistochemistry(Oxford University Press, 2018-04-01) Mortazavi, Farzad; Oblak, Adrian L.; Morrison, Will Z.; Schmahmann, Jeremy D.; Stanley, H. Eugene; Wedeen, Van J.; Rosene, Douglas L.; Pathology and Laboratory Medicine, School of MedicineBrain fiber pathways are presumed to follow smooth curves but recent high angular resolution diffusion MRI (dMRI) suggests that instead they follow 3 primary axes often nearly orthogonal. To investigate this, we analyzed axon pathways under monkey primary motor cortex with (1) dMRI tractography, (2) axon tract tracing, and (3) axon immunohistochemistry. dMRI tractography shows the predicted crossings of axons in mediolateral and dorsoventral orientations and does not show axon turns in this region. Axons labeled with tract tracer in the motor cortex dispersed in the centrum semiovale by microscopically sharp axonal turns and/or branches (radii ≤15 µm) into 2 sharply defined orientations, mediolateral and dorsoventral. Nearby sections processed with SMI-32 antibody to label projection axons and SMI-312 antibody to label all axons revealed axon distributions parallel to the tracer axons. All 3 histological methods confirmed preponderant axon distributions parallel with dMRI axes with few axons (<20%) following smooth curves or diagonal orientations. These findings indicate that axons navigate deep white matter via microscopic sharp turns and branches between primary axes. They support dMRI observations of primary fiber axes, as well as the prediction that fiber crossings include navigational events not yet directly resolved by dMRI. New methods will be needed to incorporate coherent microscopic navigation into dMRI of connectivity.Item Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research(Springer Nature, 2014-09-16) Ray, Balmiki; Chopra, Nipun; Long, Justin M.; Lahiri, Debomoy K.; Psychiatry, School of MedicineBackground: Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. Results: This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. Conclusion: We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.Item Hydrodynamic delivery for the study, treatment and prevention of acute kidney injury(2014-07-07) Corridon, Peter R.; Atkinson, Simon; Basile, David P.; Bacallao, Robert L.; Dunn, Kenneth William; Gattone II, Vincent H.Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.Item In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging(2014-02-25) Freeman, Kim Renee; Rubart-von der Lohe, Michael; Atkinson, Simon; Hurley, Thomas D., 1961-; Gattone II, Vincent H.The sympathetic nervous system strongly modulates the contractile and electrical function of the heart. The anatomical underpinnings that enable a spatially and temporally coordinated dissemination of sympathetic signals within the cardiac tissue are only incompletely characterized. In this work we took the first step of unraveling the in situ 3D microarchitecture of the cardiac sympathetic nervous system. Using a combination of two-photon excitation fluorescence microscopy and computer-assisted image analyses, we reconstructed the sympathetic network in a portion of the left ventricular epicardium from adult transgenic mice expressing a fluorescent reporter protein in all peripheral sympathetic neurons. The reconstruction revealed several organizational principles of the local sympathetic tree that synergize to enable a coordinated and efficient signal transfer to the target tissue. First, synaptic boutons are aligned with high density along much of axon-cell contacts. Second, axon segments are oriented parallel to the main, i.e., longitudinal, axes of their apposed cardiomyocytes, optimizing the frequency of transmitter release sites per axon/per cardiomyocyte. Third, the local network was partitioned into branched and/or looped sub-trees which extended both radially and tangentially through the image volume. Fourth, sub-trees arrange to not much overlap, giving rise to multiple annexed innervation domains of variable complexity and configuration. The sympathetic network in the epicardial border zone of a chronic myocardial infarction was observed to undergo substantive remodeling, which included almost complete loss of fibers at depths >10 µm from the surface, spatially heterogeneous gain of axons, irregularly shaped synaptic boutons, and formation of axonal plexuses composed of nested loops of variable length. In conclusion, we provide, to the best of our knowledge, the first in situ 3D reconstruction of the local cardiac sympathetic network in normal and injured mammalian myocardium. Mapping the sympathetic network connectivity will aid in elucidating its role in sympathetic signal transmisson and processing.Item Integrated Cytometry With Machine Learning Applied to High-Content Imaging of Human Kidney Tissue for In Situ Cell Classification and Neighborhood Analysis(Elsevier, 2023) Winfree, Seth; McNutt, Andrew T.; Khochare, Suraj; Borgard, Tyler J.; Barwinska, Daria; Sabo, Angela R.; Ferkowicz, Michael J.; Williams, James C., Jr.; Lingeman, James E.; Gulbronson, Connor J.; Kelly, Katherine J.; Sutton, Timothy A.; Dagher, Pierre C.; Eadon, Michael T.; Dunn, Kenneth W.; El-Achkar, Tarek M.; Medicine, School of MedicineThe human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.Item Large-scale, three-dimensional tissue cytometry of the human kidney: a complete and accessible pipeline(Elsevier, 2021) Ferkowicz, Michael J.; Winfree, Seth; Sabo, Angela R.; Kamocka, Malgorzata M.; Khochare, Suraj; Barwinska, Daria; Eadon, Michael T.; Cheng, Ying-Hua; Phillips, Carrie L.; Sutton, Timothy A.; Kelly, Katherine J.; Dagher, Pierre C.; El-Achkar, Tarek M.; Dunn, Kenneth W.; Kidney Precision Medicine Project; Anatomy, Cell Biology and Physiology, School of MedicineThe advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell's physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.Item Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics(American Physical Society, 2020) Tavakoli, Meysam; Jazani, Sina; Sgouralis, Ioannis; Shafraz, Omer M.; Sivasankar, Sanjeevi; Donaphon, Bryan; Levitus, Marcia; Pressé, Steve; Physics, School of ScienceFluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.Item Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues(Springer Nature, 2019-04-04) Chen, Yi; Shen, Qi; White, Sharla L.; Gokmen-Polar, Yesim; Badve, Sunil; Goodman, Laurie J.; Pathology and Laboratory Medicine, School of MedicineThe tumor microenvironment can be spatially heterogenous, which makes it challenging to fully characterize with standard 2D histology-based methods. In this study, we determined the feasibility of a CLARITY tissue-processing approach to analyze biopsies from breast cancer patients. Formalin-fixed human breast cancer core-needle biopsy specimens, were embedded, lipid-cleared, and multiplexed immunostained to identify key biomarkers (pan-cytokeratin, Ki67, CD3). Confocal microscopy was then used to image the specimens after refractive index matching. These data sets were then quantitatively compared to conventional slide-based FFPE histology. Using CLARITY, the gross and cellular morphology of the tissues were well preserved, and high optical transparency was achieved, with the exception of fibrotic regions. Specific staining of various cellular and nuclear markers was achieved using optimized antibody conditions. Manually determined composite Ki67 scores from the CLARITY datasets agreed with histology results. However, the CLARITY datasets (3D) revealed variation in the intra-tumoral Ki67 expression that was not evident in individual FFPE sections (2D). We further demonstrated that archived FFPE clinical specimens can be CLARITY-processed, immunostained, and imaged. In short, CLARITY-processed specimens may enable a more accurate, unbiased analysis of tumor samples in comparison to conventional slide-based histology, thus allowing for improved visualization of intra-tumoral heterogeneity.Item Vector Competence of Thrips Species to Transmit Soybean Vein Necrosis Virus(Frontiers, 2019-03-19) Han, Jinlong; Nalam, Vamsi J.; Yu, I-Chen; Nachappa, Punya; Anatomy and Cell Biology, IU School of MedicineSoybean vein necrosis virus (SVNV) is a newly discovered species of tospovirus infecting soybean plants that is transmitted by the primary vector, soybean thrips (Neohydatothrips variabilis), and two additional secondary vectors, tobacco thrips (Frankliniella fusca) and eastern flower thrips (F. tritici). This study was undertaken to elucidate the association between virus acquisition [6, 12, 24, and 48 h acquisition access period (AAP)] and transmission efficiency [12, 24, and 48 h inoculation access period (IAP)] in the primary vector, N. variabilis, and to examine the mechanisms of vector competence by analyzing the effect of AAP (6, 12, and 24 h) on virus infection in various tissues. In addition, we examined virus infection in tissues of the two secondary vectors. We found a significant effect of virus acquisition on transmission efficiency, transmission rate post 6 and 48 h AAP was significantly lower than 12 and 24 h AAP. Our analysis did not reveal a correlation between virus transmission rate and virus RNA in corresponding N. variabilis adults. On the contrary, N. variabilis adults harboring higher accumulation of the virus (>104) resulted in lower transmission rates. Analysis of SVNV infection in the tissues revealed the presence of the virus in the foregut, midgut (region 1, 2, and 3), tubular salivary glands and principal salivary glands (PSG) of adults of all three vector species, however, the frequency of infected tissues was highest in N. variabilis followed by F. fusca and F. tritici. The frequency of SVNV infection in individual tissues specifically the salivary glands was lowest after 6 h AAP compared to 12 and 24 h AAP. This finding is in agreement with the transmission assays, where significantly lower virus transmission rate was observed post 6 h AAP. In addition, N. variabilis adults with high PSG infection (12 and 24 h AAP) were likely to have high percentage of foregut and midgut region 2 infection. Overall, results from the transmission assays and immunolabeling experiments suggest that shorter AAP results in reduced virus infection in the various tissues especially PSG, which are important determinants of vector competence in SVNV-thrips interaction.