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Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

ABSTRACT: Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

SUBMITTER: Stadlbauer V

PROVIDER: S-EPMC7662403 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Stadlbauer Verena V Lanzerstorfer Peter P Neuhauser Cathrina C Weber Florian F Stübl Flora F Weber Petra P Wagner Michael M Plochberger Birgit B Wieser Stefan S Schneckenburger Herbert H Weghuber Julian J

International journal of molecular sciences 20201027 21

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translo ...[more]

PMID: 33120934

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Project description:Fluorescence spectroscopy and imaging are now used throughout the biosciences. Fluorescence microscopes, spectrofluorometers, microwell plate readers and microarray imagers all use multiple optical components to collect, redirect and focus the emission onto single point or array imaging detectors. For almost all biological samples, except those with regular nanoscale features, emission occurs in all directions. With the exception of complex microscope objectives with large collection angles (NA ≤ 0.5), all these instruments collect only a small fraction of the total emission. Because of the increasing knowledge base on fluorophores within near-field (<200 nm) distances from plasmonic and photonic structures we can anticipate the development of compact devices in which the sample to be detected is located directly on solid state detectors such as CCDs or CMOS cameras. Near-field interactions of fluorophores with metallic or dielectric multi-layer structures (MLSs) can capture a large fraction of the total emission. Depending on the composition and dimensions of the MLSs, the spatial distribution of the sample emission results in distinct optical patterns on the detector surface. With either plain glass slides or MLSs the most commonly used front focal plane (FFP) images reveal the x-y spatial distribution of emission from the sample. Another approach, which is often used with two or three-dimensional nanostructures, is back focal plane (BFP) imaging. The BFP images reveal the angular distribution of the emission. The FFP and BFP images occur at certain distances from the sample which is determined by the details of the optical components. Obtaining these images requires multiple optical components and distances which are too large for the compact devices. For devices described in this paper, the images will be detected at a fixed distance between the sample and some arbitrary distance below the MLS which is determined by the geometry and thicknesses of the components. We refer to measurements at these locations as out-of-focal plane (OFP) imaging. Herein we describe a method to measure the optical fields at micron and multi-micron distances below the MLS, which will represent the images seen by an optically coupled array detector. The possibility of sub-surface optical images is illustrated using five different multi-layer structures. This is accomplished using an optical configuration which allows measurement at a front focal plane (FFP), back focal plane (BFP) or any OFP locations. Our OFP imaging method provides a link between the FFP images which reveals the surface distribution of fluorophores with the BFP images that reveal the angular distribution of emission. This linkage can be useful when examining structures which have nanoscale features due to fluorescence or leakage radiation from nanostructures.

Dataset Information

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Publications

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

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