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Annual Review of Analytical Chemistry... Jun 2022Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and... (Review)
Review
Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and biomedical applications. The spectroscopic capability of SRS enables identification and tracking of specific molecules or classes of molecules, often without labeling. SRS microscopy also has the hallmark advantage of signal strength that is directly proportional to molecular concentration, allowing for in situ quantitative analysis of chemical composition of heterogeneous samples with submicron spatial resolution and subminute temporal resolution. However, it is important to recognize that quantification through SRS microscopy requires assumptions regarding both system and sample. Such assumptions are often taken axiomatically, which may lead to erroneous conclusions without proper validation. In this review, we focus on the tacitly accepted, yet complex, quantitative aspect of SRS microscopy. We discuss the various approaches to quantitative analysis, examples of such approaches, challenges in different systems, and potential solutions. Through our examination of published literature, we conclude that a scrupulous approach to experimental design can further expand the powerful and incisive quantitative capabilities of SRS microscopy.
Topics: Microscopy; Nonlinear Optical Microscopy; Spectrum Analysis, Raman
PubMed: 35300525
DOI: 10.1146/annurev-anchem-061020-015110 -
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 -
Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy.Molecules and Cells Jan 2022The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the... (Review)
Review
The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.
Topics: Microscopy, Electron; Microscopy, Fluorescence
PubMed: 35114646
DOI: 10.14348/molcells.2021.5011 -
Chimia Nov 2018The development of super-resolved optical microscopies has revolutionized the way we visualize cell biology. These techniques strongly rely on the use of photochemically... (Review)
Review
The development of super-resolved optical microscopies has revolutionized the way we visualize cell biology. These techniques strongly rely on the use of photochemically active fluorophores that display changes in their photophysical properties upon irradiation with light. Many reversible and irreversible photochemical transformations have been explored for this purpose, and different imaging techniques require specific mechanisms of photoconversion. In this review, we provide an overview of the most common strategies used for the development of fluorophores for super-resolution microscopy and give specific examples of state-of-the-art fluorogenic probes. Furthermore, we discuss their main field of application and possible directions for future developments.
Topics: Coloring Agents; Microscopy; Photochemical Processes
PubMed: 30514418
DOI: 10.2533/chimia.2018.764 -
Journal of Immunology Research 2016Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as... (Review)
Review
Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one hand in vitro live-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital, in vivo, and in situ microscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages.
Topics: DNA; Extracellular Traps; Histones; Humans; Microscopy; Microscopy, Electron; Microscopy, Fluorescence; Neutrophil Activation
PubMed: 27294157
DOI: 10.1155/2016/4604713 -
Scientific Reports Jun 2022Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in...
Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences.
Topics: Holography; Lenses; Lighting; Microscopy; Microscopy, Interference
PubMed: 35760816
DOI: 10.1038/s41598-022-14739-0 -
Nano Letters Jun 2021The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number...
The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.
Topics: Ligands; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nanoparticles
PubMed: 34125548
DOI: 10.1021/acs.nanolett.1c01666 -
Sensors (Basel, Switzerland) Jan 2022In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between... (Review)
Review
In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between light and biological matter. A common objective is to concentrate the electromagnetic energy associated with light into nanometer-scale volumes that, in many cases, can extend below the conventional Abbé diffraction limit. Dating back to the first application of surface plasmon resonance (SPR) for label-free detection of biomolecular interactions, resonant optical structures, including waveguides, ring resonators, and photonic crystals, have proven to be effective conduits for a wide range of optical enhancement effects that include enhanced excitation of photon emitters (such as quantum dots, organic dyes, and fluorescent proteins), enhanced extraction from photon emitters, enhanced optical absorption, and enhanced optical scattering (such as from Raman-scatterers and nanoparticles). The application of photonic metamaterials as a means for enhancing contrast in microscopy is a recent technological development. Through their ability to generate surface-localized and resonantly enhanced electromagnetic fields, photonic metamaterials are an effective surface for magnifying absorption, photon emission, and scattering associated with biological materials while an imaging system records spatial and temporal patterns. By replacing the conventional glass microscope slide with a photonic metamaterial, new forms of contrast and enhanced signal-to-noise are obtained for applications that include cancer diagnostics, infectious disease diagnostics, cell membrane imaging, biomolecular interaction analysis, and drug discovery. This paper will review the current state of the art in which photonic metamaterial surfaces are utilized in the context of microscopy.
Topics: Biosensing Techniques; Microscopy; Optics and Photonics; Photons; Surface Plasmon Resonance
PubMed: 35161831
DOI: 10.3390/s22031086 -
Cells Nov 2022In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the... (Review)
Review
In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.
Topics: Microscopy; Point-of-Care Systems; Lenses; Algorithms; Microscopy, Interference
PubMed: 36429102
DOI: 10.3390/cells11223670 -
Frontiers in Cellular and Infection... 2021Viruses are diverse pathogens that use host factors to enter cells and cause disease. Imaging the entry and replication phases of viruses and their interactions with... (Review)
Review
Viruses are diverse pathogens that use host factors to enter cells and cause disease. Imaging the entry and replication phases of viruses and their interactions with host factors is key to fully understanding viral infections. This review will discuss how confocal microscopy and imaging flow cytometry are used to investigate virus entry and replication mechanisms in fixed and live cells. Quantification of viral images and the use of cryo-electron microscopy to gather structural information of viruses is also explored. Using imaging to understand how viruses replicate and interact with host factors, we gain insight into cellular processes and identify novel targets to develop antiviral therapeutics and vaccines.
Topics: Cell Communication; Cryoelectron Microscopy; Flow Cytometry; Host-Pathogen Interactions; Microscopy, Confocal; Microscopy, Fluorescence; Virus Replication
PubMed: 34712624
DOI: 10.3389/fcimb.2021.749039