68 results found
Sonay AY, Kalyviotis K, Yaganoglu S, et al., 2021, Biodegradable harmonophores for targeted high-resolution in vivo tumor imaging, ACS Nano, Vol: 15, Pages: 4144-4154, ISSN: 1936-0851
Optical imaging probes have played a major role in detecting and monitoring a variety of diseases. In particular, nonlinear optical imaging probes, such as second harmonic generating (SHG) nanoprobes, hold great promise as clinical contrast agents, as they can be imaged with little background signal and unmatched long-term photostability. As their chemical composition often includes transition metals, the use of inorganic SHG nanoprobes can raise long-term health concerns. Ideally, contrast agents for biomedical applications should be degraded in vivo without any long-term toxicological consequences to the organism. Here, we developed biodegradable harmonophores (bioharmonophores) that consist of polymer-encapsulated, self-assembling peptides that generate a strong SHG signal. When functionalized with tumor cell surface markers, these reporters can target single cancer cells with high detection sensitivity in zebrafish embryos in vivo. Thus, bioharmonophores will enable an innovative approach to cancer treatment using targeted high-resolution optical imaging for diagnostics and therapy.
Welling M, Kalyviotis K, Pantazis P, 2020, Primed track: reliable volumetric single-cell tracking and lineage tracing of living specimen with dual-labeling approaches, Bio-protocol, Vol: 10, Pages: 1-10, ISSN: 2331-8325
Mammalian embryonic development starts with a single fertilized zygote that develops into a blastocyst embryo consisting of three cell types that evolve into either embryonic or extra-embryonic tissues. Lineage tracing of these cells can provide important information about the molecular and cellular dynamics contributing to fate allocation during early development. While global labeling techniques allow for visualization of all cells at the same time, lineage tracing of cells over several divisions can become complicated due to embryo movement and rotation as well as increasing cell densities. Here, we use green-to-red photoconvertible proteins for both global and sparse labeling of cells of interest in the developing murine embryo. We use primed conversion to achieve precise photoconversion of single nuclei in 4-cell stage embryos followed by volumetric live imaging to capture development up to the blastocyst stage. We developed an image analysis pipeline, called primed Track, that uses the dual labeling strategy for both straightforward segmentation and registration of all cells in the embryo as well as correction of rotational and spatial drift. Together, this strategy allows for reliable and fast tracking and lineage tracing of individual cells, even over increased imaging time intervals that result in a major reduction in data volume, all essential conditions for volumetric long-term imaging techniques
Malkinson G, Mahou P, Chaudan E, et al., 2020, Fast in vivo imaging of SHG nanoprobes with multiphoton light-sheet microscopy, ACS Photonics, Vol: 7, Pages: 1036-1049, ISSN: 2330-4022
Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO4 and BaTiO3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow.
Malkinson G, Maioli V, Boniface A, et al., 2020, Advances in fast multiphoton microscopy using light-sheet illumination
We report on recent advances in multiphoton light-sheet microscopy to perform fast multimodal imaging combining fluorescence with second-harmonic generation and to mitigate photodamage during in vivo imaging of embryos.
Kalyviotis K, Qin H, Pantazis P, 2020, Chapter 19 - PhOTO zebrafish and primed conversion: advancing the mechanistic view of development and disease, Behavioral and Neural Genetics of Zebrafish, Editors: Gerlai, Publisher: Academic Press, Pages: 309-322
A set of transgenic zebrafish lines, called PhOTO (Photoconvertible Optical Tracking Of…), were previously introduced and have wide applicability for accurate cell tracking during highly dynamic events, including embryo development, tissue regeneration, and cancer/disease progression. Cell shapes, interactions, divisions, and dynamics can be monitored by employing the photoconvertible fluorescent protein Dendra2 and the blue fluorescent protein Cerulean as compartment-specific labels of the nucleus and the plasma membrane. The combination of PhOTO zebrafish with confined primed conversion, a recently introduced axially confined photoconversion method, enables nontoxic, instantaneous, and precise cell tracking during any stage of the life cycle of zebrafish. The implementation of advanced imaging platforms and smart bioimaging software to perform high-fidelity lineage tracing of primed converted PhOTO zebrafish will greatly benefit the goal of acquiring a refined mechanistic view of development and disease.
Welling M, Mohr MA, Ponti A, et al., 2019, Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins, eLife, Vol: 8, Pages: 1-13, ISSN: 2050-084X
Accurate lineage reconstruction of mammalian pre-implantation development isessential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescencelabeling techniques is complicated by increasing cell density and rapid embryo rotation, whichhampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging datasets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (prpcFPs) to simultaneously visualize the global green and the photoconverted red population in orderto minimize tracking uncertainties over prolonged time windows. Confined primed conversion ofH2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation,classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Usinggreen and red labels as fiducial markers, we computationally correct for rotational and translationaldrift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increasedimaging time intervals – addressing major concerns in the field of volumetric embryo imaging.
Slenders E, Boye H, Urbain M, et al., 2018, Image Correlation Spectroscopy with Second Harmonic Generating Nanoparticles in Suspension and in Cells, JOURNAL OF PHYSICAL CHEMISTRY LETTERS, Vol: 9, Pages: 6112-6118, ISSN: 1948-7185
Welling M, Mohr M, Ponti A, et al., 2018, High fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins
Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions of mammalian development. Lineage tracing using global labeling techniques is complicated by increasing cell density and rapid embryo rotation, impeding automatic alignment and rendering accurate cell tracking of obtained four-dimensional imaging data sets highly challenging. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift and accomplish high fidelity lineage tracing combined with a reduced data size – addressing majors concerns in the field of volumetric embryo imaging.
Mohr MA, Pantazis P, 2018, Primed Conversion: The New Kid on the Block for Photoconversion, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 24, Pages: 8268-+, ISSN: 0947-6539
Mohr MA, Pantazis P, 2018, Frontispiece: Primed Conversion: The New Kid on the Block for Photoconversion, Chemistry - A European Journal, Vol: 24, ISSN: 0947-6539
Sugiyama N, Sonay AY, Tussiwand R, et al., 2018, Effective Labeling of Primary Somatic Stem Cells with BaTiO3 Nanocrystals for Second Harmonic Generation Imaging, Small, Vol: 14, Pages: 1-9, ISSN: 1613-6810
While nanoparticles are an increasingly popular choice for labeling and tracking stem cells in biomedical applications such as cell therapy, their intracellular fate and subsequent effect on stem cell differentiation remain elusive. To establish an effective stem cell labeling strategy, the intracellular nanocrystal concentration should be minimized to avoid adverse effects, without compromising the intensity and persistence of the signal necessary for long‐term tracking. Here, the use of second‐harmonic generating barium titanate nanocrystals is reported, whose achievable brightness allows for high contrast stem cell labeling with at least one order of magnitude lower intracellular nanocrystals than previously reported. Their long‐term photostability enables to investigate quantitatively at the single cell level their cellular fate in hematopoietic stem cells (HSCs) using both multiphoton and electron microscopy. It is found that the concentration of nanocrystals in proliferative multipotent progenitors is over 2.5‐fold greater compared to quiescent stem cells; this difference vanishes when HSCs enter a nonquiescent, proliferative state, while their potency remains unaffected. Understanding the nanoparticle stem cell interaction allows to establish an effective and safe nanoparticle labeling strategy into somatic stem cells that can critically contribute to an understanding of their in vivo therapeutic potential.
Nienhaus K, Mohr MA, Kobitski AY, et al., 2018, Primed Green-to-Red Photoconversion of Fluorescent Proteins Occurs via a Triplet State, 62nd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 533A-533A, ISSN: 0006-3495
Nugraha B, Mohr MA, Ponti A, et al., 2017, Monitoring and manipulating cellular crosstalk during kidney fibrosis inside a 3D in vitro co-culture, Scientific Reports, Vol: 7, Pages: 1-11, ISSN: 2045-2322
In pharmacological research the development of promising lead compounds requires a detailed understanding of the dynamics of disease progression. However, for many diseases, such as kidney fibrosis, gaining such understanding requires complex real-time, multi-dimensional analysis of diseased and healthy tissue. To allow for such studies with increased throughput we established a dextran hydrogel-based in vitro 3D co-culture as a disease model for kidney fibrosis aimed at the discovery of compounds modulating the epithelial/mesenchymal crosstalk. This platform mimics a simplified pathological renal microenvironment at the interface between tubular epithelial cells and surrounding quiescent fibroblasts. We combined this 3D technology with epithelial reporter cell lines expressing fluorescent biomarkers in order to visualize pathophysiological cell state changes resulting from toxin-mediated chemical injury. Epithelial cell damage onset was robustly detected by image-based monitoring, and injured epithelial spheroids induced myofibroblast differentiation of co-cultured quiescent human fibroblasts. The presented 3D co-culture system therefore provides a unique model system for screening of novel therapeutic molecules capable to interfere and modulate the dialogue between epithelial and mesenchymal cells.
Mohr MA, Kobitski AY, Sabater LR, et al., 2017, Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 56, Pages: 11628-11633, ISSN: 1433-7851
Zhang W, Lohman AW, Zhuravlova Y, et al., 2017, Optogenetic control with a photocleavable protein, PhoCl, Nature Methods, Vol: 14, Pages: 391-394, ISSN: 1548-7091
To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.
Sonay AY, Pantazis P, 2017, Bioinspired Second Harmonic Generation, Conference on Clinical and Preclinical Optical Diagnostics, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Mohr MA, Argast P, Pantazis P, 2016, Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins, NATURE PROTOCOLS, Vol: 11, Pages: 2419-2431, ISSN: 1754-2189
Welling M, Ponti A, Pantazis P, 2016, Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysis, MOLECULAR HUMAN REPRODUCTION, Vol: 22, Pages: 172-181
Mohr MA, Pantazis P, 2016, Single neuron morphology in vivo with confined primed conversion, ZEBRAFISH: CELLULAR AND DEVELOPMENTAL BIOLOGY, PT A: CELLULAR BIOLOGY, 4TH EDITION, Editors: Detrich, Westerfield, Zon, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 125-138, ISBN: 978-0-12-803475-0
Dempsey WP, Hodas NO, Ponti A, et al., 2015, Determination of the source of SHG verniers in zebrafish skeletal muscle, SCIENTIFIC REPORTS, Vol: 5, ISSN: 2045-2322
SHG microscopy is an emerging microscopic technique for medically relevant imaging because certain endogenous proteins, such as muscle myosin lattices within muscle cells, are sufficiently spatially ordered to generate detectable SHG without the use of any fluorescent dye. Given that SHG signal is sensitive to the structural state of muscle sarcomeres, SHG functional imaging can give insight into the integrity of muscle cells in vivo. Here, we report a thorough theoretical and experimental characterization of myosin-derived SHG intensity profiles within intact zebrafish skeletal muscle. We determined that “SHG vernier” patterns, regions of bifurcated SHG intensity, are illusory when sarcomeres are staggered with respect to one another. These optical artifacts arise due to the phase coherence of SHG signal generation and the Guoy phase shift of the laser at the focus. In contrast, two-photon excited fluorescence images obtained from fluorescently labeled sarcomeric components do not contain such illusory structures, regardless of the orientation of adjacent myofibers. Based on our results, we assert that complex optical artifacts such as SHG verniers should be taken into account when applying functional SHG imaging as a diagnostic readout for pathological muscle conditions.
Dempsey WP, Georgieva L, Helbling PM, et al., 2015, In vivo single-cell labeling by confined primed conversion, Nature Methods, Vol: 12, Pages: 645-648, ISSN: 1548-7091
Spatially confined green-to-red photoconversion of fluorescent proteins with high-power, pulsed laser illumination is negligible, thus precluding optical selection of single cells in vivo. We report primed conversion, in which low-power, dual-wavelength, continuous-wave illumination results in pronounced photoconversion. With a straightforward addition to a conventional confocal microscope, we show confined primed conversion in living zebrafish and reveal the complex anatomy of individual neurons packed between neighboring cells.
Pantazis P, Supatto W, 2014, Advances in whole-embryo imaging: a quantitative transition is underway, NATURE REVIEWS MOLECULAR CELL BIOLOGY, Vol: 15, Pages: 327-339, ISSN: 1471-0072
Dempsey WP, Qin H, Pantazis P, 2014, In vivo cell tracking using PhOTO zebrafish., Methods Mol Biol, Vol: 1148, Pages: 217-228
By combining the strength of previously described in vivo cell tracking methodologies, we have recently generated a set of transgenic zebrafish lines, called "PhOTO (photoconvertible optical tracking of…)" zebrafish. PhOTO zebrafish lines are suitable for cell tracking during highly dynamic events, including gastrulation, tissue regeneration, tumorigenesis, and cancer/disease progression. Global monitoring of cell shape, cell interactions, e.g., cell intercalations, coordinated division, and cell dynamics are accomplished by using fluorescence imaging of nuclear and plasma membrane fluorescent protein labeling. The irreversible green-to-red photoconversion property of Dendra2 fusions enables noninvasive, specific and high-contrast selection of targeted cells of interest, which greatly simplifies cell tracking and segmentation in time and space. Here we demonstrate photoconversion and in vivo cell tracking using PhOTO zebrafish.
Mikut R, Dickmeis T, Driever W, et al., 2013, Automated Processing of Zebrafish Imaging Data: A Survey, ZEBRAFISH, Vol: 10, Pages: 401-421, ISSN: 1545-8547
Culic-Viskota J, Dempsey WP, Fraser SE, et al., 2012, Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish, NATURE PROTOCOLS, Vol: 7, Pages: 1618-1633, ISSN: 1754-2189
Pantazis P, Bollenbach T, 2012, Transcription factor kinetics and the emerging asymmetry in the early mammalian embryo, CELL CYCLE, Vol: 11, Pages: 2055-2058, ISSN: 1538-4101
Dempsey WP, Fraser SE, Pantazis P, 2012, SHG nanoprobes: Advancing harmonic imaging in biology, BIOESSAYS, Vol: 34, Pages: 351-360, ISSN: 0265-9247
Dempsey WP, Fraser SE, Pantazis P, 2012, PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration, PLOS ONE, Vol: 7, ISSN: 1932-6203
BackgroundElucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.MethodologyPhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.Conclusions/SignificancePhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.
The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1–2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.
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