scholarly journals Storm-Lite: An Inexpensive Tool to Demonstrate Stochastic Super Resolution Microscopy Techniques in the Classroom

2017 ◽  
Vol 112 (3) ◽  
pp. 464a
Author(s):  
Anthony Wu ◽  
Lark Moreno ◽  
Maxim Prigozhin ◽  
Sharlene Denos
Nanoscale ◽  
2021 ◽  
Author(s):  
Adelaide Miranda ◽  
Ana I. Gómez-Varela ◽  
Andreas Stylianou ◽  
Liisa M. Hirvonen ◽  
Humberto Sánchez ◽  
...  

This review provides a detailed picture of the innovative efforts to combine atomic force microscopy and different super-resolution microscopy techniques to elucidate biological questions.


2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.


Author(s):  
Matthew D. Lycas ◽  
Aske L. Ejdrup ◽  
Andreas T. Sørensen ◽  
Nicolai O. Haahr ◽  
Søren H. Jørgensen ◽  
...  

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.


2021 ◽  
Author(s):  
Lisa A.E. Catsburg ◽  
Manon Westra ◽  
Annemarie M. L. van Schaik ◽  
Harold D. MacGillavry

ABSTRACTAt postsynaptic sites of neurons, a prominent clathrin-coated structure, the endocytic zone (EZ), controls the trafficking of glutamate receptors and is essential for synaptic plasticity. Despite its importance, little is known about how this clathrin structure is organized to mediate endocytosis. We used live-cell and super-resolution microscopy techniques to reveal the dynamic organization of this poorly understood clathrin structure. We found that a subset of endocytic proteins only transiently appeared at postsynaptic sites. In contrast, other proteins, including Eps15, intersectin1L, and β2-adaptin, were persistently enriched and partitioned at the edge of the EZ. We found that uncoupling the EZ from the synapse led to the loss of most of these components, while disrupting the actin cytoskeleton or AP2-membrane interactions did not alter EZ positioning. We conclude that the EZ is a stable, highly organized molecular platform where components are differentially recruited and positioned to orchestrate the endocytosis of synaptic receptors.


2020 ◽  
Author(s):  
Nicholas Hall ◽  
Josh Titlow ◽  
Martin J. Booth ◽  
Ian M. Dobbie

AbstractMicroscope-AOtools is a software package which allows for a simple, robust and generalised implementation of adaptive optics (AO) elements. It contains all the necessary methods for set-up, calibration, and aberration correction which are simple to use and function in a robust manner. Aberrations arising from sources such as sample hetero-geneity and refractive index mismatches are constant problems in biological imaging. These aberrations reduce image quality and the achievable depth of imaging, particularly in super-resolution microscopy techniques. AO technology has been proven to be effective in correcting for these aberrations and thereby improving the image quality. However, it has not been widely adopted by the biological imaging community due, in part, to difficulty in set-up and operation of AO, particularly by non-specialist users. Microscope-AOtools offers a robust, easy-to-use implementation of the essential methods for set-up and use of AO techniques. These methods are constructed in a generalised manner that can utilise a range of adaptive optics elements, wavefront sensing techniques and sensorless AO correction methods. Furthermore, the methods are designed to be easily extensible as new techniques arise, leading to a streamlined pipeline for new AO technology and techniques to be adopted by the wider microscopy community.


Author(s):  
Monika Pawlowska ◽  
Ron Tenne ◽  
Bohnishikha Ghosh ◽  
Adrian Makowski ◽  
Radek Lapkiewicz

Abstract Super-resolution microscopy techniques have pushed the limits of resolution in optical imaging by more than an order of magnitude. However, these methods often require long acquisition times as well as complex setups and sample preparation protocols. Super-resolution Optical Fluctuation Imaging (SOFI) emerged over ten years ago as an approach that exploits temporal and spatial correlations within the acquired images to obtain increased resolution with less strict requirements. This review follows the progress of SOFI from its first demonstration to the development of a branch of methods that treat fluctuations as a source of contrast, rather than noise. Among others, we highlight the implementation of SOFI with standard fluorescent proteins as well as the microscope modification that facilitate 3D imaging and the application of modern cameras. Going beyond the classical framework of SOFI, we explore different innovative concepts from deep neural networks all the way to a quantum analogue of SOFI, antibunching microscopy. While SOFI has not reached the same level of ubiquity as other super-resolution methods, our overview finds significant progress and substantial potential for the concept of leveraging fluorescence fluctuations to obtain super-resolved images.


2016 ◽  
Vol 473 (20) ◽  
pp. 3385-3399 ◽  
Author(s):  
Manuel Maidorn ◽  
Silvio O. Rizzoli ◽  
Felipe Opazo

The synapse is densely packed with proteins involved in various highly regulated processes. Synaptic protein copy numbers and their stoichiometric distribution have a drastic influence on neuronal integrity and function. Therefore, the molecular analysis of synapses is a key element to understand their architecture and function. The overall structure of the synapse has been revealed with an exquisite amount of details by electron microscopy. However, the molecular composition and the localization of proteins are more easily addressed with fluorescence imaging, especially with the improved resolution achieved by super-resolution microscopy techniques. Notably, the fast improvement of imaging instruments has not been reflected in the optimization of biological sample preparation. During recent years, large efforts have been made to generate affinity probes smaller than conventional antibodies adapted for fluorescent super-resolution imaging. In this review, we briefly discuss the current views on synaptic organization and necessary key technologies to progress in the understanding of synaptic physiology. We also highlight the challenges faced by current fluorescent super-resolution methods, and we describe the prerequisites for an ideal study of synaptic organization.


Author(s):  
Kirti Prakash ◽  
Benedict Diederich ◽  
Stefanie Reichelt ◽  
Rainer Heintzmann ◽  
Lothar Schermelleh

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.


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