scholarly journals Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

2019 ◽  
Author(s):  
Claudia Prahst ◽  
Parham Ashrafzadeh ◽  
Kyle Harrington ◽  
Lakshmi Venkatraman ◽  
Mark Richards ◽  
...  
Keyword(s):  
Mouse Models ◽  
Fluorescent Microscopy ◽  
Light Sheet ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Retinal Cell ◽  
Eye Disease ◽  
Cell Behaviour ◽  
Oxygen Induced Retinopathy ◽  
Time Lapse Imaging

AbstractAs the general population ages and the incidence of diabetes increases epidemically, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the murine eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM) and 2) LSFM readily reveals new features of even well studied eye disease mouse models, such as the Oxygen-Induced Retinopathy (OIR) model. Through correlative LSFM-Confocal imaging we find that flat-mounting retinas for confocal microscopy significantly distorts tissue morphology. The minimized distortion with LSFM dramatically improved analysis of pathological vascular tufts in the OIR model revealing “knotted” morphologies, leading to a proposed new tuft nomenclature. Furthermore, live-imaging of OIR tuft formation revealed abnormal cell motility and altered filopodia dynamics. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of pathological processes in the eye, in particular neuro-vascular degenerative processes.

scholarly journals Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Claudia Prahst ◽  
Parham Ashrafzadeh ◽  
Thomas Mead ◽  
Ana Figueiredo ◽  
Karen Chang ◽  
...  
Keyword(s):  
Mouse Models ◽  
Fluorescent Microscopy ◽  
Light Sheet ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Retinal Cell ◽  
Eye Disease ◽  
Cell Behaviour ◽  
Oxygen Induced Retinopathy ◽  
Time Lapse Imaging

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated ‘knotted’ morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neurovascular, degenerative processes.

scholarly journals Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

2021 ◽  
Author(s):  
Cassio Pedroso ◽  
Victor Mann ◽  
Kathrin Zuberbühler ◽  
Markus-Frederik Bohn ◽  
Jessica Yu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Time Lapse ◽  
Live Cell ◽  
Confocal Imaging ◽  
Cellular Targets ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Isopeptide Bonds ◽  
Time Lapse Imaging

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

scholarly journals Imaging morphogenesis

2017 ◽  
Vol 372 (1720) ◽  
pp. 20150511 ◽  
Author(s):  
Donald M. Bell
Keyword(s):  
Time Lapse ◽  
Hostile Environment ◽  
Close Attention ◽  
Cell Behaviour ◽  
Live Tissue ◽  
Repeated Observation ◽  
The Stability ◽  
Time Lapse Imaging ◽  
Informative Approach ◽  
Microscope Stage

The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of the main practical considerations to take into account when embarking on a project to image cell behaviour in the context of cells' normal surroundings. Scrutiny of these activities is likely to be the most informative approach to understanding mechanical morphogenesis but is often confounded by the substantial technical difficulties involved in imaging samples over extended periods of time. Repeated observation of cells in live tissue requires that strategies be adopted to prioritize the stability of the sample, ensuring that it remains viable and develops normally while being held in a manner accessible to microscopic examination. Key considerations when creating reliable protocols for time-lapse imaging may be broken down into three main criteria; labelling, mounting and image acquisition. Choices and compromises made here, however, will directly influence image quality, and even small refinements can substantially improve what information may be extracted from images. Live imaging of tissue is difficult but paying close attention to the basics along with a little innovation is likely to be well rewarded. This article is part of the themed issue ‘Systems morphodynamics: understanding the development of tissue hardware’.

scholarly journals Crossbill: an open access single objective light-sheet microscopy platform

2021 ◽  
Author(s):  
Manish Kumar ◽  
Sandeep Kishore ◽  
David McLean ◽  
Yevgenia Kozorovitskiy
Keyword(s):  
Open Access ◽  
Light Sheet ◽  
Time Lapse ◽  
Volumetric Imaging ◽  
Open Hardware ◽  
Light Sheet Microscopy ◽  
Multiple Alternative ◽  
Optical Configuration ◽  
Time Lapse Imaging ◽  
Single Objective

We present an open access scanned oblique plane microscopy platform Crossbill. It combines a new optical configuration, open hardware assembly, a systematic alignment protocol, and dedicated control software to provide a compact, versatile, high resolution single objective light-sheet microscopy platform. The demonstrated configuration yields the most affordable sub-micron resolution oblique plane microscopy system to date. We add galvanometer enabled tilt-invariant lateral scan for multi-plane, multi-Hz volumetric imaging capability. A precision translation stage extends stitched field of view to centimeter scale. The accompanying open software is optimized for Crossbill and can be easily extended to include alternative configurations. Using Crossbill, we demonstrate large volume structural fluorescence imaging with sub-micron lateral resolution in zebrafish and mouse brain sections. Crossbill is also capable of multiplane functional imaging, and time-lapse imaging. We suggest multiple alternative configurations to extend Crossbill to diverse microscopy applications.

scholarly journals Threshold illumination for non-invasive imaging of cells and tissues

2020 ◽  
Author(s):  
M A Bashar Emon ◽  
Samantha Knoll ◽  
Umnia Doha ◽  
Danielle Baietto ◽  
Lauren Ladehoff ◽  
...  
Keyword(s):  
Fluorescent Microscopy ◽  
Red Light ◽  
Monochromatic Light ◽  
Green Light ◽  
Time Lapse ◽  
Cultured Fibroblasts ◽  
Non Invasive ◽  
Contraction Ratio ◽  
Force Relaxation ◽  
Time Lapse Imaging

AbstractFluorescent microscopy employs monochromatic light which can affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and type of cell. In addition, we establish a threshold light that invokes minimal effect on cells. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation. The beads move towards cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio, λr, as a measure of cell response to light. The cells were exposed to green (wavelength, λ = 545-580 nm) and red (λ = 635-650 nm) light with a range of intensities. We find red light with intensity less than ~ 57 W/m2 results in λr = 1, i.e., cells maintain force homeostasis. Higher intensities and smaller wavelengths result in widespread force-relaxation in cells with λr > 1. We suggest the use of λ > 650 nm light with low intensity (I ≤ 57 W/m2) for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.

scholarly journals Enhanced in vivo-imaging in fish by optimized anaesthesia, fluorescent protein selection and removal of pigmentation

2018 ◽  
Author(s):  
Colin Q. Lischik ◽  
Leonie Adelmann ◽  
Joachim Wittbrodt
Keyword(s):  
Fluorescent Protein ◽  
Light Sheet ◽  
Time Lapse ◽  
Light Sheet Microscopy ◽  
Inner Organs ◽  
Traditional Approaches ◽  
Time Lapse Imaging ◽  
Targeted Inactivation ◽  
Imaging Conditions

AbstractFish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation.We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.

scholarly journals Author response: Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

2020 ◽  
Author(s):  
Claudia Prahst ◽  
Parham Ashrafzadeh ◽  
Thomas Mead ◽  
Ana Figueiredo ◽  
Karen Chang ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Light Sheet ◽  
Author Response ◽  
Retinal Cell ◽  
Cell Behaviour ◽  
Space And Time ◽  
Light Sheet Fluorescence Microscopy
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