A simple image processing pipeline to sharpen topology maps in multi-wavelength interference microscopy.

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps, and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed-cell specimens.

Benchmarking Thiolate-Driven Photoswitching of Cyanine Dyes.

Carbocyanines are among the best performing dyes in single-molecule localization microscopy (SMLM), but their performance critically relies on optimized photoswitching buffers. Here, we study the versatile role of thiols in cyanine photoswitching at varying intensities generated in a single acquisition by a microelectromechanical systems (MEMS) mirror placed in the excitation path. The key metrics we have analyzed as a function of the thiolate concentration are photon budget, on-state and off-state lifetimes and the corresponding impact on image resolution. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum of ∼1 mM is necessary to facilitate SMLM measurements. We also identify a concentration bandwidth of 1-16 mM in which the photoswitching performance can be balanced between high molecular brightness and high off-time to on-time ratios. Furthermore, we monitor the performance of the popular oxygen scavenger system based on glucose and glucose oxidase over time and show simple measures to avoid acidification during prolonged measurements. Finally, the impact of buffer settings is quantitatively tested on the distribution of the glucose transporter protein 4 within the plasma membrane of adipocytes. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes.

GLUT4 dispersal at the plasma membrane of adipocytes: a super-resolved journey.

In adipose tissue, insulin stimulates glucose uptake by mediating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In 2010, insulin was revealed to also have a fundamental impact on the spatial distribution of GLUT4 within the plasma membrane, with the existence of two GLUT4 populations at the plasma membrane being defined: (1) as stationary clusters and (2) as diffusible monomers. In this model, in the absence of insulin, plasma membrane-fused GLUT4 are found to behave as clusters. These clusters are thought to arise from exocytic events that retain GLUT4 at their fusion sites; this has been proposed to function as an intermediate hub between GLUT4 exocytosis and re-internalisation. By contrast, insulin stimulation induces the dispersal of GLUT4 clusters into monomers and favours a distinct type of GLUT4-vesicle fusion event, known as fusion-with-release exocytosis. Here, we review how super-resolution microscopy approaches have allowed investigation of the characteristics of plasma membrane-fused GLUT4 and further discuss regulatory step(s) involved in the GLUT4 dispersal machinery, introducing the scaffold protein EFR3 which facilitates localisation of phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) to the cell surface. We consider how dispersal may be linked to the control of transporter activity, consider whether macro-organisation may be a widely used phenomenon to control proteins within the plasma membrane, and speculate on the origin of different forms of GLUT4-vesicle exocytosis.

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton.

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

Widefield standing wave microscopy of red blood cell membrane morphology with high temporal resolution.

We report the first demonstration of widefield standing wave (SW) microscopy of fluorescently labelled red blood cells at high speeds that allow for the rapid imaging of membrane deformations. Using existing and custom MATLAB functions, we also present a method to generate 2D and 3D reconstructions of the SW data for improved visualization of the cell. We compare our technique with standard widefield epifluorescence imaging and show that the SW technique not only reveals more topographical information about the specimen but does so without increasing toxicity or the rate of photobleaching and could make this a powerful technique for the diagnosis or study of red blood cell morphology and biomechanical characteristics.