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

Browsing by Subject "Fluorescence microscopy"

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An Improved Methodology to Evaluate Cell and Molecular Signals in the Reparative Callus During Fracture Healing
(Sage, 2020-03) Kambrath, Anuradha Valiya; Williams, Justin N.; Sankar, Uma; Anatomy and Cell Biology, School of Medicine
Approximately 5% to 10% of all bone fractures do not heal completely, contributing to significant patient suffering and medical costs. Even in healthy individuals, fracture healing is associated with significant downtime and loss of productivity. However, no pharmacological treatments are currently available to promote efficient bone healing. A better understanding of the underlying molecular mechanisms is crucial for developing novel therapies to hasten healing. The early reparative callus that forms around the site of bone injury is a fragile tissue consisting of shifting cell populations held together by loose connective tissue. The delicate callus is challenging to section and is vulnerable to disintegration during the harsh steps of immunostaining, namely, decalcification, deparaffinization, and antigen retrieval. Here, we describe an improved methodology for processing early-stage fracture calluses and immunofluorescence labeling of the sections to visualize the temporal (timing) and spatial (location) patterns of cellular and molecular events that regulate bone healing. This method has a short turnaround time from sample collection to microscopy as it does not require lengthy decalcification. It preserves the structural integrity of the fragile callus as the method does not entail deparaffinization or harsh methods of antigen retrieval. Our method can be adapted for high-throughput screening of drugs that promote efficacious bone healing.
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.
Instant FLIM enables 4D in vivo lifetime imaging of intact and injured zebrafish and mouse brains
(Optica, 2021) Zhang, Yide; Guldner, Ian H.; Nichols, Evan L.; Benirschke, David; Smith, Cody J.; Zhang, Siyuan; Howard, Scott S.; Medicine, School of Medicine
Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, most existing FLIM techniques are slow to acquire and process lifetime images, difficult to implement, and expensive. Here, we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 μm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains up to 12 hours.
Large-scale annotated dataset for cochlear hair cell detection and classification
(Springer Nature, 2024-04-23) Buswinka, Christopher J.; Rosenberg, David B.; Simikyan, Rubina G.; Osgood, Richard T.; Fernandez, Katharine; Nitta, Hidetomi; Hayashi, Yushi; Liberman, Leslie W.; Nguyen, Emily; Yildiz, Erdem; Kim, Jinkyung; Jarysta, Amandine; Renauld, Justine; Wesson, Ella; Wang, Haobing; Thapa, Punam; Bordiga, Pierrick; McMurtry, Noah; Llamas, Juan; Kitcher, Siân R.; López-Porras, Ana I.; Cui, Runjia; Behnammanesh, Ghazaleh; Bird, Jonathan E.; Ballesteros, Angela; Vélez-Ortega, A. Catalina; Edge, Albert S. B.; Deans, Michael R.; Gnedeva, Ksenia; Shrestha, Brikha R.; Manor, Uri; Zhao, Bo; Ricci, Anthony J.; Tarchini, Basile; Basch, Martín L.; Stepanyan, Ruben; Landegger, Lukas D.; Rutherford, Mark A.; Liberman, M. Charles; Walters, Bradley J.; Kros, Corné J.; Richardson, Guy P.; Cunningham, Lisa L.; Indzhykulian, Artur A.; Otolaryngology -- Head and Neck Surgery, School of Medicine
Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.
Modeling and design optimization of a microfluidic chip for isolation of rare cells
(2013-12) Gannavaram, Spandana; Zhu, Likun; Yu, Huidan (Whitney); Xie, Jian; Anwar, Sohel
Cancer is still among those diseases that prominently contribute to the numerous deaths that are caused each year. But as technology and research is reaching new zeniths in the present times, cure or early detection of cancer is possible. The detection of rare cells can help understand the origin of many diseases. The current study deals with one such technology that is used for the capture or effective separation of these rare cells called Lab-on-a-chip microchip technology. The isolation and capture of rare cells is a problem uniquely suited to microfluidic devices, in which geometries on the cellular length scale can be engineered and a wide range of chemical functionalizations can be implemented. The performance of such devices is primarily affected by the chemical interaction between the cell and the capture surface and the mechanics of cell-surface collision and adhesion. This study focuses on the fundamental adhesion and transport mechanisms in rare cell-capture microdevices, and explores modern device design strategies in a transport context. The biorheology and engineering parameters of cell adhesion are defined; chip geometries are reviewed. Transport at the microscale, cell-wall interactions that result in cell motion across streamlines, is discussed. We have concentrated majorly on the fluid dynamics design of the chip. A simplified description of the device would be to say that the chip is at micro scale. There are posts arranged on the chip such that the arrangement will lead to a higher capture of rare cells. Blood consisting of rare cells will be passed through the chip and the posts will pose as an obstruction so that the interception and capture efficiency of the rare cells increases. The captured cells can be observed by fluorescence microscopy. As compared to previous studies of using solid microposts, we will be incorporating a new concept of cylindrical shell micropost. This type of micropost consists of a solid inner core and the annulus area is covered with a forest of silicon nanopillars. Utilization of such a design helps in increasing the interception and capture efficiency and reducing the hydrodynamic resistance between the cells and the posts. Computational analysis is done for different designs of the posts. Drag on the microposts due to fluid flow has a great significance on the capture efficiency of the chip. Also, the arrangement of the posts is important to contributing to the increase in the interception efficiency. The effects of these parameters on the efficiency in junction with other factors have been studied and quantified. The study is concluded by discussing design strategies with a focus on leveraging the underlying transport phenomena to maximize device performance.
Multimodal imaging reveals a unique autofluorescence signature of Randall's plaque
(Springer, 2021) Winfree, Seth; Weiler, Courtney; Bledsoe, Sharon B.; Gardner, Tony; Sommer, André J.; Evan, Andrew P.; Lingeman, James E.; Krambeck, Amy E.; Worcester, Elaine M.; El-Achkar, Tarek M.; Williams, James C., Jr.; Medicine, School of Medicine
Kidney stones frequently develop as an overgrowth on Randall's plaque (RP) which is formed in the papillary interstitium. The organic composition of RP is distinct from stone matrix in that RP contains fibrillar collagen; RP in tissue has also been shown to have two proteins that are also found in stones, but otherwise the molecular constituents of RP are unstudied. We hypothesized that RP contains unique organic molecules that can be differentiated from the stone overgrowth by fluorescence. To test this, we used micro-CT-guided polishing to expose the interior of kidney stones for multimodal imaging with multiphoton, confocal and infrared microscopy. We detected a blue autofluorescence signature unique to RP, the specificity of which was also confirmed in papillary tissue from patients with stone disease. High-resolution mineral mapping of the stone also showed a transition from the apatite within RP to the calcium oxalate in the overgrowth, demonstrating the molecular and spatial transition from the tissue to the urine. This work provides a systematic and practical approach to uncover specific fluorescence signatures which correlate with mineral type, verifies previous observations regarding mineral overgrowth onto RP and identifies a novel autofluorescence signature of RP demonstrating RP's unique molecular composition.
PIE-FLIM Measurements of Two Different FRETBased Biosensor Activities in the Same Living Cells
(Cell Press, 2020-04-21) Reissaus, Christopher A.; Day, Kathleen H.; Mirmira, Raghavendra G.; Dunn, Kenneth W.; Pavalko, Fredrick M.; Day, Richard N.; Pediatrics, School of Medicine
We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.
Probabilistic Multi-Compartment Deformable Model, Application to Cell Segmentation
(2013-07-12) Farhand, Sepehr; Tsechpenakis, Gavriil; Fang, Shiaofen; Tuceryan, Mihran
A crucial task in computer vision and biomedical image applications is to represent images in a numerically compact form for understanding, evaluating and/or mining their content. The fundamental step of this task is the segmentation of images into regions, given some homogeneity criteria, prior appearance and/or shape information criteria. Specifically, segmentation of cells in microscopic images is the first step in analyzing many biomedical applications. This thesis is a part of the project entitled "Construction and profiling of biodegradable cardiac patches for the co-delivery of bFGF and G-CSF growth factors" funded by National Institutes of Health (NIH). We present a method that simultaneously segments the population of cells while partitioning the cell regions into cytoplasm and nucleus in order to evaluate the spatial coordination on the image plane, density and orientation of cells. Having static microscopic images, with no edge information of a cytoplasm boundary and no time sequence constraints, traditional cell segmentation methods would not perform well. The proposed method combines deformable models with a probabilistic framework in a simple graphical model such that it would capture the shape, structure and appearance of a cell. The process aims at the simultaneous cell partitioning into nucleus and cytoplasm. We considered the relative topology of the two distinct cell compartments to derive a better segmentation and compensate for the lack of edge information. The framework is applied to static fluorescent microscopy, where the cultured cells are stained with calcein AM.
Remodeling of the malaria parasite and host human red cell by vesicle amplification that induces artemisinin resistance
(American Society of Hematology, 2018-03-15) Bhattacharjee, Souvik; Coppens, Isabelle; Mbengue, Alassane; Suresh, Niraja; Ghorbal, Mehdi; Slouka, Zdenek; Safeukui, Innocent; Tang, Hsin-Yao; Speicher, David W.; Stahelin, Robert V.; Mohandas, Narla; Haldar, Kasturi; Biochemistry and Molecular Biology, School of Medicine
Artemisinin resistance threatens worldwide malaria control and elimination. Elevation of phosphatidylinositol-3-phosphate (PI3P) can induce resistance in blood stages of Plasmodium falciparum The parasite unfolded protein response (UPR) has also been implicated as a proteostatic mechanism that may diminish artemisinin-induced toxic proteopathy. How PI3P acts and its connection to the UPR remain unknown, although both are conferred by mutation in P falciparum Kelch13 (K13), the marker of artemisinin resistance. Here we used cryoimmunoelectron microscopy to show that K13 concentrates at PI3P tubules/vesicles of the parasite's endoplasmic reticulum (ER) in infected red cells. K13 colocalizes and copurifies with the major virulence adhesin PfEMP1. The PfEMP1-K13 proteome is comprehensively enriched in multiple proteostasis systems of protein export, quality control, and folding in the ER and cytoplasm and UPR. Synthetic elevation of PI3P that induces resistance in absence of K13 mutation also yields signatures of proteostasis and clinical resistance. These findings imply a key role for PI3P-vesicle amplification as a mechanism of resistance of infected red cells. As validation, the major resistance mutation K13C580Y quantitatively increased PI3P tubules/vesicles, exporting them throughout the parasite and the red cell. Chemical inhibitors and fluorescence microscopy showed that alterations in PfEMP1 export to the red cell and cytoadherence of infected cells to a host endothelial receptor are features of multiple K13 mutants. Together these data suggest that amplified PI3P vesicles disseminate widespread proteostatic capacity that may neutralize artemisinins toxic proteopathy and implicate a role for the host red cell in artemisinin resistance. The mechanistic insights generated will have an impact on malaria drug development.
Segmentation of biological images containing multitarget labeling using the jelly filling framework
(SPIE, 2018-10) Gadgil, Neeraj J.; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.; Medicine, School of Medicine
Biomedical imaging when combined with digital image analysis is capable of quantitative morphological and physiological characterizations of biological structures. Recent fluorescence microscopy techniques can collect hundreds of focal plane images from deeper tissue volumes, thus enabling characterization of three-dimensional (3-D) biological structures at subcellular resolution. Automatic analysis methods are required to obtain quantitative, objective, and reproducible measurements of biological quantities. However, these images typically contain many artifacts such as poor edge details, nonuniform brightness, and distortions that vary along different axes, all of which complicate the automatic image analysis. Another challenge is due to "multitarget labeling," in which a single probe labels multiple biological entities in acquired images. We present a "jelly filling" method for segmentation of 3-D biological images containing multitarget labeling. Intuitively, our iterative segmentation method is based on filling disjoint tubule regions of an image with a jelly-like fluid. This helps in the detection of components that are "floating" within a labeled jelly. Experimental results show that our proposed method is effective in segmenting important biological quantities.

Browsing by Subject "Fluorescence microscopy"

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