Dataset Information

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

ABSTRACT: Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10?minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9?min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

SUBMITTER: Maioli V

PROVIDER: S-EPMC5122899 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

Maioli Vincent V Chennell George G Sparks Hugh H Lana Tobia T Kumar Sunil S Carling David D Sardini Alessandro A Dunsby Chris C

Scientific reports 20161125

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor ...[more]

PMID: 27886235

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Project description:Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumor compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Spheroids are often only formed from a single cell type which does not represent the situation in vivo. However, despite this, both spheroids and organoids can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course. To address this, we have developed an R Shiny app called SpheroidAnalyseR: a simple, fast, effective open-source app that allows the analysis of spheroid or organoid size data generated in a 96-well format. SpheroidAnalyseR processes and analyzes datasets of image measurements that can be obtained via a bespoke software, described herein, that automates spheroid imaging and quantification using the Nikon A1R Confocal Laser Scanning Microscope. However, templates are provided to enable users to input spheroid image measurements obtained by user-preferred methods. SpheroidAnalyseR facilitates outlier identification and removal followed by graphical visualization of spheroid measurements across multiple predefined parameters such as time, cell-type and treatment(s). Spheroid imaging and analysis can, thus, be reduced from hours to minutes, removing the requirement for substantial manual data manipulation in a spreadsheet application. The combination of spheroid generation in 96-well ultra-low attachment microplates, imaging using our bespoke software, and analysis using SpheroidAnalyseR toolkit allows high throughput, longitudinal quantification of 3D spheroid growth whilst minimizing user input and significantly improving the efficiency and reproducibility of data analysis. Our bespoke imaging software is available from https://github.com/GliomaGenomics. SpheroidAnalyseR is available at https://spheroidanalyser.leeds.ac.uk, and the source code found at https://github.com/GliomaGenomics.

Dataset Information

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

Publications

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

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