Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores

• Published in: International journal of molecular sciences 2020-10, Vol.21 (19), p.7099
• Main Authors: Richter, Verena, Lanzerstorfer, Peter, Weghuber, Julian, Schneckenburger, Herbert
• Format: Article
• Language: English
• Subjects: Animals; Aperture; Aromatic compounds; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell Survival; Chemical compounds; Chemical properties; CHO Cells; Cricetulus; Dogs; Fluorescence; fluorescence imaging; Fluorophores; Glucose; Glucose transporter; Glucose Transporter Type 4 - analysis; Glucose Transporter Type 4 - metabolism; Green fluorescent protein; Humans; Hypoglycemic Agents - pharmacology; Illumination; Imaging, Three-Dimensional - methods; Insulin; Insulin - pharmacology; Intracellular; Lasers; Light; Localization; Luminescent Agents - analysis; Luminescent Agents - metabolism; Luminescent Proteins - analysis; Luminescent Proteins - metabolism; Madin Darby Canine Kidney Cells; Membrane vesicles; Membranes; Microenvironments; Microscopy; Microscopy, Fluorescence - methods; Mimetic compounds; Open source software; Optical Imaging - methods; Plasma; Protein Transport - drug effects; Red Fluorescent Protein; SIM; Software; Spatial light modulators; super-resolution microscopy; TIRF
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21197099

Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1-2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment.