High-speed 3-D fluorescence imaging by oblique plane microscopy: multi-well plate-reader development, biological applications and image analysis

Maioli, Vincent

doi:10.25560/68022

Cited by 3 publications

(2 citation statements)

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“…Light-sheet microscopy has the benefits that the illumination light is used to excite multiple points in the sample along its propagation direction and that a whole 2D image can be acquired in parallel. Previous attempts to characterize the spatial variation in calcium transient development across the cell in 2D using the single-objective-type LSFM method of OPM were limited by cell contraction and low SNR ( Maioli, 2016 ). Compared to this previous work, the dual-objective flexible LSFM system has higher detection NA and detection efficiency, which enables higher spatio-temporal resolution for the same light dose or a lower laser power for equivalent spatio-temporal sampling.…”

Section: Discussionmentioning

confidence: 99%

“…Spark detection and characterization was performed using a custom MATLAB code adapted from an earlier version implemented for the detection and characterization of sparks in OPM 2D time-lapse data ( Maioli, 2016 ; Sikkel et al, 2016 ). Calcium spark analysis was performed on the last 5,000 frames (12.7 s) of each acquisition during which the electrical stimulation was switched off.…”

Section: Methodsmentioning

confidence: 99%

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High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes

et al. 2023

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Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity.Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 μm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell.Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules.Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes

et al. 2023

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A Versatile Oblique Plane Microscope for Large-Scale and High-Resolution Imaging of Subcellular Dynamics

Sapoznik

Chang

Huh

et al. 2020

Preprint

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We present a single-objective light-sheet microscope, also known as an oblique-plane microscope, that uses a bespoke glass-tipped tertiary objective and improves the resolution, field of view, usability, and stability over previous variants. Owing to its high numerical aperture optics, this microscope achieves the highest lateral resolution in light-sheet fluorescence microscopy, and its axial resolution is similar to that of Lattice Light-Sheet Microscopy. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and natural killer cell-mediated cell death. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through a confined space within a microfluidic device, photoactivation of PI3K, and diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz.

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Environmentally dependent and independent control of cell shape determination by Rho GTPase regulators in melanoma

Curry

Sparks

Bousgouni

et al. 2021

Preprint

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In order to invade 3D tissues, cancer cells dynamically change cell morphology in response to geometric and mechanical cues in the environment. But how cells determine their shape in 3D versus 2D environments is poorly understood. Studying 2D versus 3D single cell shape determination has historically been technically difficult due to the lack of methodologies to directly compare the two environments. We developed an approach to study cell shape in 2D versus 3D by measuring cell shape at different depths in collagen using stage-scanning oblique plane microscopy (ssOPM). We find characteristic shape changes occur in melanoma cells depending on whether a cell is attached to a 2D surface or 3D environment, and that these changes can be modulated by Rho GTPase regulatory proteins. Our data suggest that regulation of cell protrusivity undergoes a switch of control between different Rho GTPase regulators depending on the physical microenvironment.

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High-speed 3-D fluorescence imaging by oblique plane microscopy: multi-well plate-reader development, biological applications and image analysis

Cited by 3 publications

References 0 publications

High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes

High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes

A Versatile Oblique Plane Microscope for Large-Scale and High-Resolution Imaging of Subcellular Dynamics

Environmentally dependent and independent control of cell shape determination by Rho GTPase regulators in melanoma

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