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Expert Review of Hematology May 2017Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often... (Review)
Review
Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy - the so-called 'super-resolution' microscopies - as a possible solution. Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application. Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.
Topics: Blood Platelets; Gray Platelet Syndrome; Humans; Microscopy, Fluorescence
PubMed: 28374619
DOI: 10.1080/17474086.2017.1315302 -
Philosophical Transactions of the Royal... May 2017Systems morphodynamics describes a multi-level analysis of mechanical morphogenesis that draws on new microscopy and computational technologies and embraces a systems...
Systems morphodynamics describes a multi-level analysis of mechanical morphogenesis that draws on new microscopy and computational technologies and embraces a systems biology-informed scope. We present a selection of articles that illustrate and explain this rapidly progressing field.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
Topics: Microscopy; Morphogenesis; Systems Biology
PubMed: 28348260
DOI: 10.1098/rstb.2016.0505 -
Current Opinion in Structural Biology Feb 2021Protein organization modification plays a vital role in initiating signaling pathways, transcriptional regulation, and cell apoptosis regulation. Simultaneous... (Review)
Review
Protein organization modification plays a vital role in initiating signaling pathways, transcriptional regulation, and cell apoptosis regulation. Simultaneous quantification of oligomeric state and cellular parameters in the same cell, even though challenging, is required to understand their correlation at the molecular level. Recent advances of fluorescence protein and single-molecule localization microscopy enables the determination of localizations and oligomeric states of target proteins in cells. We reviewed the fluorescence intensity-based, localization-based, and photophysical property-based approaches for in-cell quantification of protein oligomeric stoichiometry. We discussed their working principles, applications, advantages, and limitations. These results also imply the combination of methodologies targeting different biological parameters at the single-cell level is essential to uncover the structure-function relationship at the molecular level.
Topics: Microscopy, Fluorescence; Proteins; Single Molecule Imaging
PubMed: 33242727
DOI: 10.1016/j.sbi.2020.10.022 -
Journal of Structural Biology Feb 2017Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare...
Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.
Topics: Cryoelectron Microscopy; Electron Microscope Tomography; Microscopy; Microscopy, Fluorescence; Software
PubMed: 27368127
DOI: 10.1016/j.jsb.2016.06.020 -
Archives of Pathology & Laboratory... Feb 2011Advances in microscopy enable visualization of a broad range of new morphologic features. (Review)
Review
CONTEXT
Advances in microscopy enable visualization of a broad range of new morphologic features.
OBJECTIVE
To review and illustrate advances in microscopy with relevance to pathologists.
DATA SOURCES
Literature review and new observations.
RESULTS
Fluorescence microscopy enables multiantigen detection; allows novel optical-sectioning techniques, with some advantages compared to paraffin sectioning; and permits live-cell imaging. Live-cell imaging allows pathologists to move from a period when all diagnostic expertise was reliant on interpreting static images to a period when cellular dynamics can play a role in diagnosis. New techniques have bypassed by about 100-fold what had long been believed to be a limit to the resolution of light microscopy. Fluorescence resonance energy transfer (FRET) appears capable of visualizing diagnostically relevant molecular events in living or fixed cells that are immeasurable by other molecular techniques. We describe applications of 2-photon microscopy, FRET, structured illumination, and the subdiffraction techniques of near-field microscopy, photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy.
CONCLUSION
New microscopy techniques present opportunities for pathologists to develop improved diagnostic tests.
Topics: Animals; Humans; Microscopy; Pathology
PubMed: 21284447
DOI: 10.5858/135.2.255 -
Biochemical Society Transactions Dec 2023Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution... (Review)
Review
Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.
Topics: Cryoelectron Microscopy; Microscopy, Fluorescence; Microscopy, Electron; Cold Temperature
PubMed: 38015555
DOI: 10.1042/BST20221246 -
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 -
Science Progress 2012Robert Hooke was a polymath whose expertise during the 17th century spanned many different scientific areas. As a schoolboy on the Isle of Wight he was obsessed with the...
Robert Hooke was a polymath whose expertise during the 17th century spanned many different scientific areas. As a schoolboy on the Isle of Wight he was obsessed with the possibility of human flight and later became equally absorbed in cosmology and planetary motion. His skills as an artist were put to good use both as an architect following the Great Fire of London and before that in Micrographia. Although that book is best known for demonstrating the power of Hooke's microscope, Micrographia describes distant planetary bodies, the wave theory of light, the organic origin of fossils, and various other philosophical and scientific interests of its author The following thumbnail sketches of Hooke reveal him to be a man of enormous energy and imagination whose ideas were often pirated or under-rated.
Topics: Architecture; Astronomy; History, 17th Century; History, 18th Century; Microscopy; Paleontology; United Kingdom
PubMed: 23094324
DOI: 10.3184/003685012X13454653990042 -
The Journal of Neuroscience : the... Oct 2018In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell... (Review)
Review
In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting.
Topics: Animals; Brain; Humans; Imaging, Three-Dimensional; Microscopy; Microscopy, Confocal; Microscopy, Fluorescence; Nerve Net; Neurons
PubMed: 30381424
DOI: 10.1523/JNEUROSCI.1677-18.2018 -
Nature Protocols Feb 2021The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled...
The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated 3D resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and analyze the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a troubleshooting guide and present examples of anticipated results. An experienced optical instrument builder will require ~12 months from the start of ordering hardware components to acquiring high-quality biological images.
Topics: Humans; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 33328610
DOI: 10.1038/s41596-020-00428-7