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Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sarah E. Webb, Jeffrey J. Kelu, Andrew L. Miller
In one series of experiments, SMCs dissected from embryos at 48 hpf were dual-immunolabelled with antibodies to TPC2 and RyR (or to IP3R type III and RyR) for subsequent dual-colour stimulated emission depletion (STED) super-resolution microscopy (Hell and Wichmann, 1994). In these experiments, the immunolabelling procedure was exactly the same as that used for the other zebrafish cell culture labelling experiments, using Atto 647N-tagged and Alexa Fluor 488-tagged secondary antibodies. These 647 nm and 488 nm dyes have distinct excitation and emission spectra and both have been reported to demonstrate good STED efficiency (Nishimune et al., 2016; Willig et al., 2007). Dual-colour STED imaging demonstrated that there were “nano-gaps” of ~50–90 nm between TPC2 clusters and distinct RyR punctae. As this distance was within a hypothetical limit (Fameli et al., 2014), this suggests that Ca2+ released via TPC2 might act as a trigger to stimulate Ca2+-induced Ca2+ release in the SR via activation of the RyR (Kelu et al., 2017; Morgan et al., 2013). Unfortunately, TPC2 and IP3R type III could not be dual-immunolabelled as these antibodies were both raised in rabbits. However, when the RyR and IP3R type III were dual-immunolabelled and imaged via dual-STED, no distinct gaps between the two were observed.
Detectors, Relative Dosimetry, and Microdosimetry
Published in Harald Paganetti, Proton Therapy Physics, 2018
For spatial microdosimetry, it is of course important that the spatial resolution of the detector system is sufficient to resolve individual ionizations or ionization clusters. This is usually achieved in low-density gas environments, such as in a cloud chamber, but can also be achieved in solids in combination with confocal or super resolution microscopy to visualize the reaction products from ionization clusters.
Introduction to optical imaging
Published in Ahmad Fadzil Mohamad Hani, Dileep Kumar, Optical Imaging for Biomedical and Clinical Applications, 2017
Dileep Kumar, Ahmad Fadzil Mohamad Hani
STED [30] is one of the most widely used microscopic techniques used to obtain high-resolution microscopic images called as super resolution microscopy. Super resolution microscopy uses STED in order to create subdiffraction limit features that are obtained by altering the effective point spread function of the excitation pulse using a second laser suppressing fluorescence emission through fluorophores placed at a distant location from the center of excitation. Before the spontaneous emission occurs, the molecule is sent back to ground state by the stimulated emission. This stimulated emission is obtained with the use of a two overlapping synchronised laser beams, which arrive one after the other with the time gap of less than the lifetime of molecule excited. The second pulse is of doughnut shape in order to reduce the spreading of the pulse and make the scanning of sample sharper. The pulses are shown in Figure 1.11. STED produces sharper and high-resolution images than that of confocal microscopy.
Use of electron microscopy to study platelets and thrombi
Published in Platelets, 2020
Maurizio Tomaiuolo, Rustem I. Litvinov, John W. Weisel, Timothy J. Stalker
Newer light microscopy techniques, collectively referred to as super resolution microscopy, have achieved resolutions breaking the limits of light diffraction. These approaches are particularly useful for determining subcellular localization and/or colocalization of proteins within cells and even intracellular organelles such as platelet α-granules [93]. However, with resolution limits in the 20–100 nm XY range [94], super resolution microscopy still does not achieve the single nanometer resolution possible using EM. Thus, rather than supplant EM, super resolution microscopy remains a complementary imaging modality. In fact, fluorescence microscopy (including super resolution techniques) may now be combined with EM, an approach referred to as correlative light and electron microscopy (CLEM). Further, EM approaches continue to advance as new sample preparation techniques and microscope technologies become available. For example, various chemical fixation methods are known to poorly preserve membrane structures and have the potential to introduce artifacts such as membrane blebbing [95]. Application of high-pressure freezing with freeze substitution protocols for fixation/dehydration, as well as vitrification of isolated platelets directly on EM grids, has opened new opportunities to examine platelet internal membranes in near native states (e.g. cryo-EM). Finally, multiple approaches for the examination of cells and tissues at EM resolution in 3-dimensions have been developed, including electron tomography (ET), serial block face SEM (SBF-SEM) and focused ion beam SEM (FIB-SEM). Each of these newer EM approaches overcomes specific limitations of conventional EM approaches, while maintaining EM level resolution. Advantages and limitations of each are described in the sections below.
Bridging the gap: Super-resolution microscopy of epithelial cell junctions
Published in Tissue Barriers, 2018
Emily I. Bartle, Tejeshwar C. Rao, Tara M. Urner, Alexa L. Mattheyses
Advancements in microscopy have bridged this gap by combining the advantages of fluorescence techniques with nanoscale resolution. Super-resolution microscopy overcomes the diffraction barrier, allowing imaging of fluorescently labeled samples at resolutions from 120 nm, double the resolution of conventional microscopy, down to 20 nm in the x-y plane.11-14 These revolutionary techniques allow for the study of nanoscale organization of protein arrangement, order, and dynamics in macromolecular complexes.
Seeing is believing: use of advanced imaging to study platelets and megakaryocytes
Published in Platelets, 2020
Steven G. Thomas, Natalie S. Poulter
It is often said that a picture is worth a thousand words. Whether you believe this or not, being able to see a cellular structure at high resolution, or a biological process occurring in real time, certainly has many benefits in terms of understanding the function and relevance of the cells and pathways that we study. Imaging of platelets and megakaryocytes has come a long way since simple light microscopes were used in the late nineteenth century by Max Schultze, who was the first to identify platelets (1865), and Giulio Bizzozero, who first coined the name ‘platelets’ and demonstrated their role in hemostasis and thrombosis (1882) (reviewed in [1]). These men viewed their unstained samples on simple light microscopes and their observations were therefore restricted to high contrast, morphological features. In 1906 James Wright was the first person to discover that platelets were derived from the bone marrow cell the megakaryocyte, by examining stained histological sections, again using a simple light microscope [2]. Whereas these early studies were performed on simple instruments, in more recent years technological advances, not just in the design of the microscope optics, but also in methods for preparing and labeling cells, mean we now have a myriad of ways in which to ‘see’ cells. The development of fluorescence microscopy began a modern microscopy revolution by allowing us to label and track multiple proteins in living cells and therefore to measure movement and interactions in real time [3,4]. In the last 10 years, super-resolution microscopy has taken this further as it combines fluorescence labeling of proteins with clever tricks to circumvent the diffraction limit of light to increase resolution down to tens of nm, allowing unprecedented details of the inner workings of a cell to be visualized [5]. Electron microscopy, first developed in the 1930s, has the highest resolution of all and provides structural details of cells that proved vital in the advances of cellular biology in the 1950s and 60s [6]. It has also undergone a resurgence in recent years and can now be combined with fluorescence techniques in correlative methods giving unprecedented levels of structural and functional detail [7].