Standing wave microscopy of red blood cell membrane morphology with high temporal resolution

Widefield fluorescence microscopy is an integral tool for life science imaging though the achievable resolutions are limited by the diffraction nature of light. One technique to increase the axial resolution is known as standing wave microscopy [1]. The standing wave can be generated by placing a mirror at the specimen plane which causes interference between the incoming and reflected excitation illumination. The axial resolution is reduced to λ/4n as only fluorophores which are in the location of the full width at the half maximum of the antinodes are excited [2] resulting in periodic bands of fluorescence

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 an exocytic event that retains GLUT4 at the fusion site; 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 favors 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. [Abstract copyright: Copyright 2023 The Author(s).

Optimizing photoswitching performance of organic dyes for SMLM through a single MEMS mirror

Whilst SMLM is able to localize molecules with nanometre precision it is only able to achieve this if the imaging parameters have been properly optimised. Key parameters we have investigated for optimisation are homogeneous excitation illumination and the optimal pH and thiol concentrations for photoswitching buffers. Typical SMLM experiments make use of conventional gaussian illumination modes meaning either a compromise in the excitation intensity is made due to overfilling of the objective lens, or an uneven illumination field of view (FOV) is observed which can cause intensity driven photoswitching differences in dye molecules located at different points in the FOV. We demonstrate the use of a single microelectromechanical system (MEMS) mirror as a cost-effective method to generate a flat-field of illumination across the FOV resulting in consistent SMLM metrics. We also show a workflow employing an intensity gradient through the MEMS in which we screen for optimal pH and thiol concentrations to obtain the best results for brightness and photoswitching performances of the carbocyanine dye Alexa Fluor 647. Finally, we have monitored the performance of the oxygen scavenger system based on glucose and glucose oxidase in open or closed environments, determining the amount of acidification present in prolonged imaging experiments

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 micro-electromechanical systems (MEMS) mirror placed in the excitation path. The key metrics we have analysed 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

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 multiwavelength 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

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

A photoacoustic microscopy system using MEMS and fibre tip transducers for all-optical control

A 3D photoacoustic microscopy (PAM) system is presented and characterized, using optical MEMS and fibre tip transducers as active elements to provide all-optical positioning and read-out. The excitation beam position is controlled using an electrostatically actuated 2-axis MEMS scanner. This allows for fast 3D scanning without motion-induced artefacts caused by stage scanning, and selective imaging of regions of interest. A 20 MHz fibre tip transducer is used for acoustic detection, which allows a variety of sample holders to be used including common approaches such as multi-well plates and petri-dishes

3D-printing optical components for microscopy using a desktop 3D-printer

In this work, we present the results of using a commercially available SLA printer for the fabrication of a range of designs of optical components. The optical properties are compared to off-the-shelf optics, including a detailed analysis of optical transmission, part uniformity and surface quality. A post-processing refinement step is introduced whose results are benchmarked against off-the-shelf polished glass lenses to exemplify sub-hundred nanometre surface roughness uniformity with minimal surface defects, and transmission properties as high as 85% at 638 nm for a 1 mm thick optical block without anti-reflection coatings

A structured illumination microscopy module using two micro-electromechanical system scanning micromirrors

We present the development and application of a novel structured illumination microscope (SIM) in which the grating pattern is generated using two optical beams controlled via two micro-electro-mechanical system (MEMS) 3D scanning micromirrors, each having static angular and piston control. This arrangement enables the generation of a fully controllable spatial interference pattern at the focal plane by adjusting the positions of the beams in the back-aperture of a high numerical aperture (NA) microscope objective. The utilization of MEMS micromirrors to control angular, radial and phase positioning for the structured illumination patterns has advantages of flexible control of the fluorescence excitation illumination, with achromatic beam delivery through the same optical path, reduced spatial footprint and cost-efficient integration

Physical activity-academic achievement: student and teacher perspectives on the 'new' nexus

Background: The association between physical activity/fitness with cognitive and academic functioning has become a topic of considerable research interest. Increasingly, schooling systems are being expected to respond to these relationships through curricular and extra-curricular interventions.Purpose: This paper reports on the qualitative findings of the impact of the Active Kids Active Minds (AKAM) intervention that included one hour of moderate to vigorous daily physical activity for the promotion of learning in a regional primary school in Australia. It gives student and teacher voice to the corpus of literature on physical activity and academic performance intervention studies that are gaining momentum in the bid to justify and promote forms of school-based physical activity.Participants: Twelve Year 5 students, their classroom teachers, and the school principal's perspectives are shared in this paper. They were key informants from 107 students and 5 teachers who participated in the intervention.Data collection: Students, their classroom teachers, and the school principal were interviewed individually or in groups by a member of the research team. Researcher field observations, along with a diary kept by the dedicated AKAM teacher, were used to interrogate the complexity and pragmatics of both delivering the intervention and succeeding in the intervention.Data analysis: Transcribed interviews were reviewed independently by the authors for recurring themes. Field observations and the AKAM teacher diary were used to triangulate interview data.Findings: Data suggested that the intervention group benefited from and welcomed the additional daily physical activity when it offered high time-on-task, fun, and reflected students' interests. The intervention design with a dedicated physical activity leader and professional development support seemingly promoted teachers' confidence and enthusiasm.Conclusions: While this intervention was designed to complement physical education, we raise questions about how physical activity in schools may be channelled towards a new wave of instrumental outcomes. © 201