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Ultramicroscopy Aug 2020In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field...
In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy. CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided. Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm.
Topics: Bacterial Proteins; Carbocyanines; Equipment Design; Fluorescence; Gold Colloid; Human Umbilical Vein Endothelial Cells; Humans; Luminescent Proteins; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 32470633
DOI: 10.1016/j.ultramic.2020.113007 -
Annual Review of Biophysics May 2023Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade,... (Review)
Review
Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade, complementing X-ray crystallography and nuclear magnetic resonance. Constant methodological improvements in both cryo-EM hardware and image processing software continue to contribute to an exponential growth in the number of structures solved annually. In this review, we provide a historical view of the many steps that were required to make cryo-EM a successful method for the determination of high-resolution protein complex structures. We further discuss aspects of cryo-EM methodology that are the greatest pitfalls challenging successful structure determination to date. Lastly, we highlight and propose potential future developments that would improve the method even further in the near future.
Topics: Cryoelectron Microscopy; Electrons; Single Molecule Imaging
PubMed: 37159297
DOI: 10.1146/annurev-biophys-111622-091300 -
Journal of Physics. Condensed Matter :... Jul 2017Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited... (Review)
Review
Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.
Topics: Cell Membrane; Microscopy; Microscopy, Confocal; Microscopy, Fluorescence
PubMed: 28481213
DOI: 10.1088/1361-648X/aa7185 -
Annual Review of Biochemistry Jun 2021The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds,... (Review)
Review
The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.
Topics: Cryoelectron Microscopy; Crystallization; Drug Discovery; Electrons; Microscopy, Electron, Transmission; Nanoparticles; Proteins; Workflow
PubMed: 34153215
DOI: 10.1146/annurev-biochem-081720-020121 -
Research in Microbiology Nov 2018The visualization of viral particles only became possible after the advent of the electron microscope. The first bacteriophage images were published in 1940 and were... (Review)
Review
The visualization of viral particles only became possible after the advent of the electron microscope. The first bacteriophage images were published in 1940 and were soon followed by many other publications that helped to elucidate the structure of the particles and their interaction with the bacterial hosts. As sample preparation improved and new technologies were developed, phage imaging became important approach to morphologically classify these viruses and helped to understand its importance in the biosphere. In this review we discuss the main milestones in phage imaging, how it affected our knowledge on these viruses and recent developments in the field.
Topics: Animals; Bacteria; Bacteriophages; History, 20th Century; History, 21st Century; Humans; Microscopy; Molecular Imaging; Virion
PubMed: 29852217
DOI: 10.1016/j.resmic.2018.05.006 -
BMC Biology Jun 2017Expansion microscopy (ExM) is a recently invented technology that uses swellable charged polymers, synthesized densely and with appropriate topology throughout a...
Expansion microscopy (ExM) is a recently invented technology that uses swellable charged polymers, synthesized densely and with appropriate topology throughout a preserved biological specimen, to physically magnify the specimen 100-fold in volume, or more, in an isotropic fashion. ExM enables nanoscale resolution imaging of preserved samples on inexpensive, fast, conventional microscopes. How does ExM work? How good is its performance? How do you get going on using it? In this Q&A, we provide the answers to these and other questions about this new and rapidly spreading toolbox.
Topics: Microscopy; Microscopy, Fluorescence; Polymers; Specimen Handling
PubMed: 28629474
DOI: 10.1186/s12915-017-0393-3 -
Current Opinion in Genetics &... Oct 2011The recently developed Coherent Anti-stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy have provided new methods to visualize... (Review)
Review
The recently developed Coherent Anti-stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy have provided new methods to visualize the localization and regulation of biological molecules without the use of invasive and potentially perturbative labels. They allow rapid imaging of specific molecules with high resolution and sensitivity. These tools have been effectively applied to the study of lipid metabolism using Caenorhabditis elegans as a genetic model, unraveling new lipid storage phenotypes and their regulatory mechanisms. Here we review the underlying principle of CARS and SRS microscopy, as well as their recent applications in lipid biology research in C. elegans.
Topics: Animals; Humans; Lipid Metabolism; Lipids; Microscopy; Spectrum Analysis, Raman
PubMed: 21945002
DOI: 10.1016/j.gde.2011.09.003 -
Philosophical Transactions of the Royal... May 2017The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of... (Review)
Review
The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of the main practical considerations to take into account when embarking on a project to image cell behaviour in the context of cells' normal surroundings. Scrutiny of these activities is likely to be the most informative approach to understanding mechanical morphogenesis but is often confounded by the substantial technical difficulties involved in imaging samples over extended periods of time. Repeated observation of cells in live tissue requires that strategies be adopted to prioritize the stability of the sample, ensuring that it remains viable and develops normally while being held in a manner accessible to microscopic examination. Key considerations when creating reliable protocols for time-lapse imaging may be broken down into three main criteria; labelling, mounting and image acquisition. Choices and compromises made here, however, will directly influence image quality, and even small refinements can substantially improve what information may be extracted from images. Live imaging of tissue is difficult but paying close attention to the basics along with a little innovation is likely to be well rewarded.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
Topics: Microscopy; Morphogenesis; Time-Lapse Imaging
PubMed: 28348248
DOI: 10.1098/rstb.2015.0511 -
Viruses Feb 2021Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to... (Review)
Review
Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to study plant virus-host interactions, making it possible to have major advances in the knowledge of plant virus infection cycles. The imaging techniques used to study plant-virus interactions have included light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopies. Here, we review the use of these techniques in plant virology, illustrating recent advances in the area with examples from plant virus replication and virus plant-to-plant vertical transmission processes.
Topics: Animals; Host Microbial Interactions; Infectious Disease Transmission, Vertical; Microscopy, Confocal; Plant Diseases; Plant Viruses; Plants; Virus Replication
PubMed: 33668729
DOI: 10.3390/v13030358 -
Neurobiology of Disease Jul 2021One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in... (Review)
Review
One of the biggest unsolved questions in neuroscience is how molecules and neuronal circuitry create behaviors, and how their misregulation or dysfunction results in neurological disease. Light microscopy is a vital tool for the study of neural molecules and circuits. However, the fundamental optical diffraction limit precludes the use of conventional light microscopy for sufficient characterization of critical signaling compartments and nanoscopic organizations of synapse-associated molecules. We have witnessed rapid development of super-resolution microscopy methods that circumvent the resolution limit by controlling the number of emitting molecules in specific imaging volumes and allow highly resolved imaging in the 10-100 nm range. Most recently, Expansion Microscopy (ExM) emerged as an alternative solution to overcome the diffraction limit by physically magnifying biological specimens, including nervous systems. Here, we discuss how ExM works in general and currently available ExM methods. We then review ExM imaging in a wide range of nervous systems, including Caenorhabditis elegans, Drosophila, zebrafish, mouse, and human, and their applications to synaptic imaging, neuronal tracing, and the study of neurological disease. Finally, we provide our prospects for expansion microscopy as a powerful nanoscale imaging tool in the neurosciences.
Topics: Animals; Brain; Brain Chemistry; Humans; Microscopy; Microscopy, Fluorescence; Nanotechnology; Neurosciences; Synapses
PubMed: 33813047
DOI: 10.1016/j.nbd.2021.105362