Tools and limitations to study the molecular composition of synapses by fluorescence microscopy

2016 ◽  
Vol 473 (20) ◽  
pp. 3385-3399 ◽  
Author(s):  
Manuel Maidorn ◽  
Silvio O. Rizzoli ◽  
Felipe Opazo
Keyword(s):  
Super Resolution ◽  
Molecular Composition ◽  
Synaptic Organization ◽  
Synaptic Physiology ◽  
Copy Numbers ◽  
Resolution Imaging ◽  
And Function ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Affinity Probes

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.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

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.

scholarly journals Microscope-AOtools: A generalised adaptive optics implementation

2020 ◽  
Author(s):  
Nicholas Hall ◽  
Josh Titlow ◽  
Martin J. Booth ◽  
Ian M. Dobbie
Keyword(s):  
Image Quality ◽  
Adaptive Optics ◽  
Super Resolution ◽  
Biological Imaging ◽  
New Techniques ◽  
Reduce Image Quality ◽  
Set Up ◽  
And Function ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

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.

scholarly journals A new probe for super-resolution imaging of membranes elucidates trafficking pathways

2014 ◽  
Vol 205 (4) ◽  
pp. 591-606 ◽  
Author(s):  
Natalia H. Revelo ◽  
Dirk Kamin ◽  
Sven Truckenbrodt ◽  
Aaron B. Wong ◽  
Kirsten Reuter-Jessen ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Cultured Cells ◽  
Neuromuscular Junctions ◽  
Organ Of Corti ◽  
Super Resolution ◽  
Molecular Composition ◽  
Cultured Neurons ◽  
Vesicle Recycling ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.

scholarly journals Super-resolution structured illumination microscopy: past, present and future

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kirti Prakash ◽  
Benedict Diederich ◽  
Stefanie Reichelt ◽  
Rainer Heintzmann ◽  
Lothar Schermelleh
Keyword(s):  
Three Dimensional ◽  
Super Resolution ◽  
Computational Imaging ◽  
Biological Applications ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Recent Developments ◽  
Resolution Imaging ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

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)’.

scholarly journals Super-resolution imaging to reveal the nanostructure of tripartite synapses

2021 ◽  
Author(s):  
Natalija Aleksejenko ◽  
Janosch Peter Heller
Keyword(s):  
Electron Microscopy ◽  
Signal Transduction ◽  
Signal Transmission ◽  
Super Resolution ◽  
Cell Types ◽  
Resolution Imaging ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Flow Of Information ◽  
The Brain

Even though neurons are the main drivers of information processing in the brain and spinal cord, other cell types are important to mediate adequate flow of information. These include electrically-passive glial cells such as microglia and astrocytes, which recently emerged as active partners facilitating proper signal transduction. In disease, these cells undergo pathophysiological changes that propel disease progression and change synaptic connections and signal transmission. In the healthy brain, astrocytic processes contact pre- and postsynaptic structures. These processes can be nanoscopic, and therefore only electron microscopy has been able to reveal their structure and morphology. However, electron microscopy is not suitable in revealing dynamic changes, and it is labour- and time-intensive. The dawn of super-resolution microscopy, techniques that ‘break’ the diffraction limit of conventional light microscopy, over the last decades has enabled researchers to reveal the nanoscopic synaptic environment. In this review, we highlight and discuss recent advances in our understanding of the nano-world of the so-called tripartite synapses, the relationship between pre- and postsynapse as well as astrocytic processes. Overall, novel super-resolution microscopy methods are needed to fully illuminate the intimate relationship between glia and neuronal cells that underlies signal transduction in the brain and that might be affected in diseases such as Alzheimer’s disease and epilepsy.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

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
Keyword(s):  
Super Resolution ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

scholarly journals The architecture and operating mechanism of a cnidarian stinging organelle

2021 ◽  
Author(s):  
Matthew Gibson ◽  
Ahmet Karabulut ◽  
Melainia McClain ◽  
Boris Rubinstein ◽  
Sean McKinney
Keyword(s):  
Structural Complexity ◽  
Super Resolution ◽  
Operating Mechanism ◽  
Sea Anemones ◽  
Form And Function ◽  
3D Electron Microscopy ◽  
Genetic Perturbations ◽  
Resolution Imaging ◽  
The Operating Mechanism ◽  
And Function

Abstract The stingers of jellyfish, sea anemones and other cnidarians, known as nematocysts, are remarkable cellular weapons used for both predation and defense1. Nematocysts are specialized organelles which consist of a pressurized capsule containing a coiled harpoon-like thread2. These structures are in turn built within specialized cells known as nematocytes3. When triggered4, the capsule explosively discharges, ejecting the coiled thread which punctures the target and rapidly elongates by turning inside out in a process called eversion5,6. Due to the structural complexity of the thread and the extreme speed of discharge, the precise mechanics of nematocyst firing have remained elusive7. Here, using a combination of live and super-resolution imaging, 3D electron microscopy and genetic perturbations, we define the step-by-step sequence of nematocyst operation in the model sea anemone Nematostella vectensis. This analysis reveals the complex biomechanical transformations underpinning the operating mechanism of nematocysts, one of the nature’s most exquisite biological micro-machines. Further, this study will provide insight into the form and function of related cnidarian organelles and serve as a template for the design of bioinspired microdevices.

scholarly journals How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

Nanoscale ◽  
2021 ◽  
Author(s):  
Adelaide Miranda ◽  
Ana I. Gómez-Varela ◽  
Andreas Stylianou ◽  
Liisa M. Hirvonen ◽  
Humberto Sánchez ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Super Resolution ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Detailed Picture

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.

scholarly journals Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10023-10033 ◽  
Author(s):  
Jan Bergstrand ◽  
Lei Xu ◽  
Xinyan Miao ◽  
Nailin Li ◽  
Ozan Öktem ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dictionary Learning ◽  
Super Resolution ◽  
Distribution Patterns ◽  
Specific Protein ◽  
Protein Distribution ◽  
Activated Platelets ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution imaging of P-selectin in platelets together with dictionary learning allow specifically activated platelets to be identified in an automatic objective manner.

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