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Journal of Structural Biology Sep 2022Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent...
Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength, etc., which influences the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77 K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.
Topics: Cold Temperature; Cryoelectron Microscopy; Electrons; Microscopy, Electron; Microscopy, Fluorescence
PubMed: 35811036
DOI: 10.1016/j.jsb.2022.107881 -
Archives of Pathology & Laboratory... Nov 2010Digital viewing of histologic images is moving from presentations and publications to incorporation into the daily work of practicing pathologists. Many technologic... (Review)
Review
CONTEXT
Digital viewing of histologic images is moving from presentations and publications to incorporation into the daily work of practicing pathologists. Many technologic limitations have been overcome recently, which should make widespread adoption more practical. The task now is for pathologists to become actively involved in its development and implementation, to ensure that the technology is developed with the intent to optimize workflow and to maintain diagnostic accuracy. An understanding of the basic precepts of digital imaging is required to make informed decisions related to hardware and software implementation and to collaborate with vendors and professionals outside of pathology (eg, regulatory agencies) as the technology rapidly develops.
OBJECTIVE
To describe the state of digital microscopy as it applies to the field of pathology and to define specific issues related to adoption of whole slide imaging systems.
DATA SOURCES
The information is derived from the experience of the author and review of the literature.
CONCLUSIONS
Digital microscopy is an important tool for surgical pathologists. It is currently an area of intense and rapid technologic development that will likely transform the workflow of many laboratories during the next several years.
Topics: Image Processing, Computer-Assisted; Laboratories; Microscopy; Pathology, Surgical
PubMed: 21043820
DOI: 10.5858/2009-0579-RAR1.1 -
Biosensors Oct 2021Microscopy is a traditional method to perform ex vivo/in vitro dental research. Contemporary microscopic techniques offer the opportunity to observe dental tissues and... (Review)
Review
Microscopy is a traditional method to perform ex vivo/in vitro dental research. Contemporary microscopic techniques offer the opportunity to observe dental tissues and materials up to nanoscale level. The aim of this paper was to perform a literature review on four microscopic methods, which are widely employed in dental studies concerning the evaluation of resin-dental adhesive interfaces-confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The literature search was performed using digital databases: PubMed, Web of Science and Scopus. On the basis of key words relevant to the topic and established eligibility criteria, finally 84 papers were included in the review. Presented microscopic techniques differ in their principle of operation and require specific protocols for specimen preparation. With regard to adhesion studies, microscopy assists in the description of several elements involved in adhesive bonding, as well as in the assessment of the condition of enamel surface and the most appropriate etching procedures. There are several factors determining the quality of the interaction between the substrates which could be recognized and a potential for further implementation of microscopic techniques in dental research could be recognized, especially when these techniques are used simultaneously or combined with spectroscopic methods. Through such microscopy techniques it is possible to provide clinically relevant conclusions and recommendations, which can be easily introduced for enamel-safe bonding and bonding protocols, as well as optimal pretreatments in dentine preparation.
Topics: Dental Bonding; Dentin; Microscopy, Atomic Force; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Resin Cements; Specimen Handling; Surface Properties
PubMed: 34821624
DOI: 10.3390/bios11110408 -
IEEE Reviews in Biomedical Engineering 2016Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and... (Review)
Review
Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.
Topics: Cell Nucleus; Image Interpretation, Computer-Assisted; Microscopy
PubMed: 26742143
DOI: 10.1109/RBME.2016.2515127 -
Developmental Cell Dec 2012It is a challenge in developmental biology to understand how an embryo's genes, proteins, and cells function and interact to govern morphogenesis, cell fate... (Review)
Review
It is a challenge in developmental biology to understand how an embryo's genes, proteins, and cells function and interact to govern morphogenesis, cell fate specification, and patterning. These processes span very different spatial and temporal scales. Despite much progress, simultaneous observation of such vastly differing scales has been beyond the scope of conventional microscopy. Light sheet microscopy fills this gap and is increasingly used for long-term, high-speed recordings of large specimens with high contrast and up to subcellular spatial resolution. We provide an overview of applications of light sheet microscopy in developmental biology and discuss future perspectives in this field.
Topics: Animals; Cell Communication; Developmental Biology; Embryonic Development; Gene Expression; Microscopy; Microscopy, Confocal
PubMed: 23237945
DOI: 10.1016/j.devcel.2012.10.008 -
Chemical Reviews Nov 2021Self-assembly is promising for construction of a wide variety of supramolecular assemblies, whose 1D/2D/3D structures are typically relevant to their functions. In-depth... (Review)
Review
Self-assembly is promising for construction of a wide variety of supramolecular assemblies, whose 1D/2D/3D structures are typically relevant to their functions. In-depth understanding of their structure-function relationships is essential for rational design and development of functional molecular assemblies. Microscopic imaging has been used as a powerful tool to elucidate structures of individual molecular assemblies with subnanometer to millimeter resolution, which is complementary to conventional spectroscopic techniques that provide the ensemble structural information. In this review, we highlight the representative examples of visualization of molecular assemblies by use of electron microscopy, atomic force microscopy, confocal microscopy, and super-resolution microscopy. This review comprehensively describes imaging of supramolecular nanofibers/gels, micelles/vesicles, coacervate droplets, polymer assemblies, and protein/DNA assemblies. Advanced imaging techniques that can address key challenges, like evaluation of dynamics of molecular assemblies, multicomponent self-assembly, and self-assembly/disassembly in complex cellular milieu, are also discussed. We believe this review would provide guidelines for deeper structural analyses of molecular assemblies to develop the next-generation materials.
Topics: Electrons; Micelles; Microscopy, Atomic Force; Microscopy, Confocal; Nanofibers
PubMed: 33942610
DOI: 10.1021/acs.chemrev.0c01334 -
Microscopy Research and Technique Dec 2020Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoVs), causative of major outbreaks in the past two decades, has claimed many lives all over the world. The virus... (Review)
Review
Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoVs), causative of major outbreaks in the past two decades, has claimed many lives all over the world. The virus effectively spreads through saliva aerosols or nasal discharge from an infected person. Currently, no specific vaccines or treatments exist for coronavirus; however, several attempts are being made to develop possible treatments. Hence, it is important to study the viral structure and life cycle to understand its functionality, activity, and infectious nature. Further, such studies can aid in the development of vaccinations against this virus. Microscopy plays an important role in examining the structure and topology of the virus as well as pathogenesis in infected host cells. This review deals with different microscopy techniques including electron microscopy, atomic force microscopy, fluorescence microscopy as well as computational methods to elucidate various prospects of this life-threatening virus.
Topics: Animals; Chlorocebus aethiops; Computational Biology; Coronavirus Infections; Host-Pathogen Interactions; Humans; Microscopy; Microscopy, Atomic Force; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Severe acute respiratory syndrome-related coronavirus; Spike Glycoprotein, Coronavirus; Vero Cells
PubMed: 32770582
DOI: 10.1002/jemt.23551 -
Communications Biology Mar 2024For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological... (Review)
Review
For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies.
Topics: Animals; Microscopy, Fluorescence, Multiphoton; Microscopy, Fluorescence; Intravital Microscopy; Spectrum Analysis; Photons
PubMed: 38531976
DOI: 10.1038/s42003-024-06057-0 -
International Journal of Molecular... Sep 2013Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies... (Review)
Review
Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.
Topics: Microscopy; Microscopy, Polarization
PubMed: 24022688
DOI: 10.3390/ijms140918520 -
Traffic (Copenhagen, Denmark) Jan 2002Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field... (Comparative Study)
Comparative Study Review
Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.
Topics: Animals; Computational Biology; Databases as Topic; Microscopy, Confocal; Microscopy, Fluorescence; Photons; Software
PubMed: 11872140
DOI: 10.1034/j.1600-0854.2002.30105.x