Browsing by Subject "Fluorescence"

Now showing 1 - 10 of 11
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    A pan-cancer organoid platform for precision medicine
    (Elsevier, 2021) Larsen, Brian M.; Kannan, Madhavi; Langer, Lee F.; Leibowitz, Benjamin D.; Bentaieb, Aicha; Cancino, Andrea; Dolgalev, Igor; Drummond, Bridgette E.; Dry, Jonathan R.; Ho, Chi-Sing; Khullar, Gaurav; Krantz, Benjamin A.; Mapes, Brandon; McKinnon, Kelly E.; Metti, Jessica; Perera, Jason F.; Rand, Tim A.; Sanchez-Freire, Veronica; Shaxted, Jenna M.; Stein, Michelle M.; Streit, Michael A.; Tan, Yi-Hung Carol; Zhang, Yilin; Zhao, Ende; Venkataraman, Jagadish; Stumpe, Martin C.; Borgia, Jeffrey A.; Masood, Ashiq; Catenacci, Daniel V. T.; Mathews, Jeremy V.; Gursel, Demirkan B.; Wei, Jian-Jun; Welling, Theodore H.; Simeone, Diane M.; White, Kevin P.; Khan, Aly A.; Igartua, Catherine; Salahudeen, Ameen A.; Medicine, School of Medicine
    Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.
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    Deep Tissue Fluorescent Imaging in Scattering Specimens Using Confocal Microscopy
    (Cambridge University Press, 2011-08) Clendenon, Sherry G.; Young, Pamela A.; Ferkowicz, Michael; Phillips, Carrie; Dunn, Kenneth W.; Department of Pediatrics, IU School of Medicine
    In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging.
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    Distinct Chemical Determinants are Essential for Achieving Ligands for Superior Optical Detection of Specific Amyloid-β Deposits in Alzheimer's Disease
    (Wiley, 2024) Wu, Xiongyu; Shirani, Hamid; Vidal, Ruben; Ghetti, Bernardino; Ingelsson, Martin; Klingstedt, Therése; Nilsson, K. Peter R.; Pathology and Laboratory Medicine, School of Medicine
    Aggregated forms of different proteins are common hallmarks for several neurodegenerative diseases, including Alzheimer's disease, and ligands that selectively detect specific protein aggregates are vital. Herein, we investigate the molecular requirements of thiophene-vinyl-benzothiazole based ligands to detect a specific type of Aβ deposits found in individuals with dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. The staining of these Aβ deposits was alternated when switching the terminal heterocyclic moiety attached to the thiophene-vinyl-benzothiazole scaffold. The most prevalent staining was observed for ligands having a terminal 3-methyl-1H-indazole moiety or a terminal 1,2-dimethoxybenzene moiety, verifying that specific molecular interactions between these ligands and the aggregates were necessary. The synthesis of additional thiophene-vinyl-benzothiazole ligands aided in pinpointing additional crucial chemical determinants, such as positioning of nitrogen atoms and methyl substituents, for achieving optimal staining of Aβ aggregates. When combining the optimized thiophene-vinyl-benzothiazole based ligands with a conventional ligand, CN-PiB, distinct staining patterns were observed for sporadic Alzheimer's disease versus dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. Our findings provide chemical insights for developing novel ligands that allow for a more precise assignment of Aβ deposits, and might also aid in creating novel agents for clinical imaging of distinct Aβ aggregates in AD.
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    Dual-ligand fluorescence microscopy enables chronological and spatial histological assignment of distinct amyloid-β deposits
    (Elsevier, 2025) Klingstedt, Therése; Shirani, Hamid; Parvin, Farjana; Nyström, Sofie; Hammarström, Per; Graff, Caroline; Ingelsson, Martin; Vidal, Ruben; Ghetti, Bernardino; Sehlin, Dag; Syvänen, Stina; Nilsson, K. Peter. R.; Pathology and Laboratory Medicine, School of Medicine
    Different types of deposits comprised of amyloid-β (Aβ) peptides are one of the pathological hallmarks of Alzheimer's disease (AD) and novel methods that enable identification of a diversity of Aβ deposits during the AD continuum are essential for understanding the role of these aggregates during the pathogenesis. Herein, different combinations of five fluorescent thiophene-based ligands were used for detection of Aβ deposits in brain tissue sections from transgenic mouse models with aggregated Aβ pathology, as well as brain tissue sections from patients affected by sporadic or dominantly inherited AD. When analyzing the sections with fluorescence microscopy, distinct ligand staining patterns related to the transgenic mouse model or to the age of the mice were observed. Likewise, specific staining patterns of different Aβ deposits were revealed for sporadic versus dominantly inherited AD, as well as for distinct brain regions in sporadic AD. Thus, by using dual-staining protocols with multiple combinations of fluorescent ligands, a chronological and spatial histological designation of different Aβ deposits could be achieved. This study demonstrates the potential of our approach for resolving the role and presence of distinct Aβ aggregates during the AD continuum and pinpoints the necessity of using multiple ligands to obtain an accurate assignment of different Aβ deposits in the neuropathological evaluation of AD, as well as when evaluating therapeutic strategies targeting Aβ aggregates.
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    Luminescence-Based MicroRNA Detection Methods
    (2012-08-27) Cissell, Kyle A.; Deo, Sapna K.; Long, Eric C. (Eric Charles); Simpson, Garth; Mao, Chengde
    MicroRNAs (miRNA) are short, 18-24 nucleotide long noncoding RNAs. These small RNAs, which are initially transcribed in the nucleus, are transported into the cell cytoplasm where they regulate protein translation either through direct cleavage of mRNA, or indirect inhibition through binding to mRNA and disrupting the protein translation machinery. Recently, miRNAs have gained much attention due to their implication in numerous diseases and cancers. It has been found that heightened or lowered levels of miRNA in diseased cells vs. healthy cells are linked to disease progression. It is therefore immensely important to be able to detect these small molecules. Current detection methods of Northern blotting, microarrays, and qRT-PCR suffer from drawbacks including low sensitivity, a lack of simplicity, being semi-quantitative in nature, time-consuming, and requiring expensive instruments. This work aims to develop novel miRNA technologies which will address these above problems. Bioluminescent labels are promising alternatives to current methods of miRNA detection. Bioluminescent labels are relatively small, similar in size to fluorescent proteins, and they emit very intense signals upon binding to their substrate. Bioluminescent labels are advantageous to fluorescent labels in that they do not require an external excitation source, rather, the excitation energy is supplied through a biochemical reaction. Therefore, background signal due to excitation is eliminated. They also have the advantage of being produced in large amounts through bacterial expression. Four miRNA detection methods are presented which utilize luminescence-based methods. Three employ Renilla luciferase, a bioluminescent protein, and one is based on fluorescence. The presented methods are capable of detecting miRNA from the picomole (nanomolar) level down to the femtomole (picomolar) level. These methods are rapid, sensitive, simple, and quantitative, can be employed in complex matrices, and do not require expensive instruments. All methods are hybridization-based and do not require amplification steps.
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    Monitoring focal adhesion kinase phosphorylation dynamics in live cells
    (Royal Society of Chemistry, 2017-07-24) Damayanti, Nur P.; Buno, Kevin; Narayanan, Nagarajan; Harbin, Sherry L Voytik; Deng, Meng; Irudayaraj, Joseph M.K.; Medicine, School of Medicine
    Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.
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    Stochastic approach to the molecular counting problem in superresolution microscopy.
    (PNAS, 2015-01-13) Rollins, Geoffrey C.; Yen Shin, Jae; Bustamante, Carlos; Pressé, Steve; Department of Physics, School of Science
    Superresolution imaging methods--now widely used to characterize biological structures below the diffraction limit--are poised to reveal in quantitative detail the stoichiometry of protein complexes in living cells. In practice, the photophysical properties of the fluorophores used as tags in superresolution methods have posed a severe theoretical challenge toward achieving this goal. Here we develop a stochastic approach to enumerate fluorophores in a diffraction-limited area measured by superresolution microscopy. The method is a generalization of aggregated Markov methods developed in the ion channel literature for studying gating dynamics. We show that the method accurately and precisely enumerates fluorophores in simulated data while simultaneously determining the kinetic rates that govern the stochastic photophysics of the fluorophores to improve the prediction's accuracy. This stochastic method overcomes several critical limitations of temporal thresholding methods.
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    Thiophene-Based Ligands for Specific Assignment of Distinct Aβ Pathologies in Alzheimer's Disease
    (American Chemical Society, 2024) Klingstedt, Therése; Lantz, Linda; Shirani, Hamid; Ge, Junyue; Hanrieder, Jörg; Vidal, Ruben; Ghetti, Bernardino; Nilsson, K. Peter R.; Pathology and Laboratory Medicine, School of Medicine
    Aggregated species of amyloid-β (Aβ) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different Aβ deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of Aβ deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to Aβ deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive Aβ plaques, whereas LL-1 mainly stained cored and neuritic Aβ deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of Aβ plaques. The ligand-labeled Aβ deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the Aβ aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain Aβ peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse Aβ deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated Aβ species associated with different forms of AD.
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    Thiophene‐Based Optical Ligands That Selectively Detect Aβ Pathology in Alzheimer's Disease
    (Wiley, 2021-08-03) Klingstedt, Therése; Shirani, Hamid; Ghetti, Bernardino; Nilsson, K. Peter R.; Pathology and Laboratory Medicine, School of Medicine
    In several neurodegenerative diseases, the presence of aggregates of specific proteins in the brain is a significant pathological hallmark; thus, developing ligands able to bind to the aggregated proteins is essential for any effort related to imaging and therapeutics. Here we report the synthesis of thiophene‐based ligands containing nitrogen heterocycles. The ligands selectively recognized amyloid‐β (Aβ) aggregates in brain tissue from individuals diagnosed neuropathologically as having Alzheimer's disease (AD). The selectivity for Aβ was dependent on the position of nitrogen in the heterocyclic compounds, and the ability to bind Aβ was shown to be reduced when introducing anionic substituents on the thiophene backbone. Our findings provide the structural and functional basis for the development of ligands that can differentiate between aggregated proteinaceous species comprised of distinct proteins. These ligands might also be powerful tools for studying the pathogenesis of Aβ aggregation and for designing molecules for imaging of Aβ pathology.
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    Using FLIM-FRET for Characterizing Spatial Interactions in the Spindle
    (Springer, 2022) Ems-McClung, Stephanie C.; Walczak, Claire E.; Biochemistry and Molecular Biology, School of Medicine
    Proper spindle assembly and the attachment of chromosomes to the spindle are key for the accurate segregation of chromosomes to daughter cells. Errors in these processes can lead to aneuploidy, which is a hallmark of cancer. Understanding the mechanisms that drive spindle assembly will provide fundamental insights into how accurate chromosome segregation is achieved. One challenge in elucidating the complexities of spindle assembly is to visualize protein interactions in space and time. The Xenopus egg extract system has been a valuable tool to probe protein function during spindle assembly in vitro. Tagging proteins with fluorescent proteins and utilizing fluorescence-based approaches, such as Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), have provided visual clues about the mechanics of spindle assembly and its regulators. However, elucidating how spindle assembly factors are spatially regulated is still challenging. Combining the egg extract system and visual FRET approaches provides a powerful tool to probe the processes involved in spindle assembly. Here we describe how a FLIM-FRET biosensor can be used to study protein-protein interactions in spindles assembled in Xenopus egg extracts. This approach should be readily adaptable to a wide variety of proteins to allow for new insights into the regulation of spindle assembly.