Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores

Abstract

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.

Impact of Doxorubicin on Cell-Substrate Topology

“…However, with a view to validate our method, some open questions remain to be resolved: are there any additional effects due to the retention time of the cells in culture medium (without any drug), e.g., due to stiffening of microtubules [ 26 ] or cellular traction forces [ 27 ]? Previous findings of CHO-K1 cells expressing a human insulin receptor (hIR) and glucose transporter 4-myc-GFP [ 28 ] proved that growth time of the cells in a culture medium may have an impact on cell-substrate topology. In the present case, prolongation of the cultivation time from 72 h to 96 h did not change the cell-substrate distances, and after incubation of the cells for 2 h with doxorubicin, these distances were only slightly, but not significantly, larger if a growth time of 96 h instead of 72 h was chosen.…”

Fluorescence microscopy-based quantitation of GLUT4 translocation

“…After incubation to facilitate endocytosis, single molecules could be followed by live cell imaging, allowing for the analysis of intracellular GLUT4 dynamics in different compartments and upon insulin stimulation at a single-molecule resolution [61]. The speed at which stained GLUT4 Microscopy approaches for a quantitative analysis of GLUT4 translocation (B): Transmission electron microscopy (TEM) in which a beam of electrons is transmitted through a specimen to form an image, which bases on the interaction of the electrons with the sample as the beam is transmitted through the specimen [56,57], confocal microscopy which increases optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation [28], total internal reflection (TIRF) microscopy which allows imaging of fluorescent molecules located close to the glass/specimen) interface by employing an evanescent wave for excitation of the fluorophores instead of direct illumination via light delivered [58], and various techniques that allow images to have resolutions higher than those imposed by the diffraction limit, including structured illumination microscopy (SIM)-TIRF [59], sample thinning enhanced resolution microscopy (STERM) [60], stimulated emission depletion (STED) microscopy [61,62], fluorescence photoactivation localization microscopy (FPALM)-TIRF [63], direct stochastic optical reconstruction microscopy (dSTORM)-TIRF [64].…”

“…During SIM, pictures are recorded with different phases and rotation angles, and using a Fourier transformation, a superresolution image can be calculated; however, SIM does not enable single-molecule detection and tracking. SIM was used to study the translocation of myc-GLUT4-GFP from intracellular vesicles (detected by SIM) to the plasma membrane (measured by TIRF-SIM) in live cells [59,88].…”

Recent Progress in the Correlative Structured Illumination Microscopy

“…Second, there is the acquisition time for each individual raw image. Compared with increasing the fluorescence lifetime of the sample, researchers are generally more enthusiastic about the possibility of improving the system performance to achieve fast illumination switching [74][75][76][77][78].…”