1,721,482 research outputs found
High dynamic range fluorescence imaging
No full textFluorescence acquisition and image display over a high dynamic range is highly desirable. However, the limited dynamic range of current photodetectors and imaging charge-coupled devices impose a limit on the fluorescence intensities that can be simultaneously captured during a single image acquisition. This is particularly troublesome when imaging biological samples, where protein expression fluctuates considerably. As a result, biological images will often contain regions with signal that is either saturated or hidden within background noise, causing information loss. In this paper, we summarize recent work from our group and others, to extended conventional to high dynamic range fluorescence imaging. These strategies have many biological applications, such as mapping of neural connections, vascular imaging, biodistribution studies or pharmacologic imaging at the single cell and organ level
Two-Photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement
No full textSmall molecule therapeutic drugs must reach their intended cellular targets(pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics).These processes are often difficult to measure in vivo due to their complexities andoccurrence within single cells. It has been particularly difficult to directly measure cellulardrug target binding
Improved intravital microscopy via synchronization of respiration and holder stabilization
No full textA major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy
Advanced Motion Compensation Methods for Intravital Optical Microscopy
No full textIntravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions
A Systems Approach for Tumor Pharmacokinetics
No full textRecent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.National Institutes of Health (U.S.) (grant T32 CA079443
Effective treatment of systemic candidiasis by synergistic targeting of cell wall synthesis
No full textFungal infections pose a serious threat to global human health fueled by the increase in immunosuppressive therapies, medical implants, and transplantation. The emergence of multidrug resistance with limited options of current antifungal drugs are a further constraint. There is thus a clear and unmet need to identify therapeutic targets and develop alternative classes of antifungal agents. Here, we hypothesize that dual targeting of key regulatory genes of fungal cell wall synthesis (FKS1 encoding beta-1,3-glucan synthase and CHS3 encoding chitin synthase) can synergistically inhibit fungal growth. Based on iterative designs, we generate a small library of fungal-targeted nanoconstructs, and identify a lead construct (FTNx) that shows preferential accumulation in fungal cells over mammalian cells and leads to prominent antifungal effects in vitro. We further show that FTNx is highly effective in a mouse model of disseminated candidiasis, demonstrating diminished fungal growth and enhanced survival rate. This strategy appears promising as an effective treatment for fungal infections in mammalian hosts.
Development of a Bioorthogonal and Highly Efficient Conjugation Method for Quantum Dots using Tetrazine-Norbornene Cycloaddition
No full textWe present a bioorthogonal and modular conjugation method for efficient coupling of organic dyes and biomolecules to quantum dots (QDs) using a norbornene−tetrazine cycloaddition. The use of noncoordinating functional groups combined with the rapid rate of the cycloaddition leads to highly efficient conjugation. We have applied this method to the in situ targeting of norbornene-coated QDs to live cancer cells labeled with tetrazine-modified proteins.National Institutes of Health (U.S.) (NIH Grant 5-U54-CA119349-05)National Institutes of Health (U.S.) (NIH Grant 5R01CA126642-02)National Institutes of Health (U.S.) (NIH Grant U01-HL080731)National Institutes of Health (U.S.) (NIH Grant T32-CA79443
Fluorescence anisotropy imaging in drug discovery
No full textNon-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/fluorescence anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting have proven difficult until recently. With the advent of high-resolution fluorescence anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital fluorescence anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs
WNT5A/JNK and FGF/MAPK Pathways Regulate the Cellular Events Shaping the Vertebrate Limb Bud
No full textSummaryBackgroundThe vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown.ResultsWe show that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, we demonstrate that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. We show that WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb.ConclusionsTogether, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development
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