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Chemical Society Reviews Jul 2020Single molecules can now be visualised with unprecedented precision. As the resolution of single-molecule experiments improves, so too does the breadth, quantity and... (Review)
Review
Single molecules can now be visualised with unprecedented precision. As the resolution of single-molecule experiments improves, so too does the breadth, quantity and quality of information that can be extracted using these methodologies. In the field of DNA nanotechnology, we use programmable interactions between nucleic acids to generate complex, multidimensional structures. We can use single-molecule techniques - ranging from electron and fluorescence microscopies to electrical and force spectroscopies - to report on the structure, morphology, robustness, sample heterogeneity and other properties of these DNA nanoconstructs. In this Tutorial Review, we will detail how complementarity between static and dynamic single-molecule techniques can provide a unified image of DNA nanoarchitectures. The single-molecule methods that we discuss provide unprecedented insight into chemical and structural behaviour, yielding not just an average outcome but reporting on the distribution of values, ultimately showing how bulk properties arise from the collective behaviour of individual structures. As the fields of both DNA nanotechnology and single-molecule characterisation intertwine, a feedback loop is generated between disciplines, providing new opportunities for the development and operation of DNA-based materials as sensors, delivery vehicles, machinery and structural scaffolds.
Topics: Biosensing Techniques; DNA; Microscopy, Atomic Force; Microscopy, Electron; Microscopy, Fluorescence; Nanostructures; Nucleic Acid Conformation; Single Molecule Imaging
PubMed: 32538403
DOI: 10.1039/c9cs00776h -
Microscopy Research and Technique Aug 2023Imaging is one of the key technologies underpinning discoveries in biomedical research. Each imaging technique however usually only provides a specific type of... (Review)
Review
Imaging is one of the key technologies underpinning discoveries in biomedical research. Each imaging technique however usually only provides a specific type of information. For instance, live-cell imaging using fluorescent tags can show us the dynamics of a system. On the other hand, electron microscopy (EM) gives us better resolution combined with the structural reference space. By applying a combination of light and electron microscopy modalities to a single sample one can exploit the advantages of both techniques in correlative light electron microscopy (CLEM). Although CLEM approaches can generate additional insights into the sample that cannot be gained by either technique in isolation, the visualization of the object of interest via markers or probes is still one of the bottlenecks in a Correlative Microscopy workflow. Whereas fluorescence is not directly visible in a standard electron microscope, gold particles, as the most common choice of probe for EM can also only be visualized using specialized light microscopes. In this review we will discuss some of the latest developments of probes for CLEM and some strategies how to choose a probe, discussing pros and cons of specific probes, and ensuring that they function as a dual modality marker.
Topics: Electrons; Microscopy, Electron; Microscopy, Fluorescence
PubMed: 36846978
DOI: 10.1002/jemt.24304 -
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 -
Microscopy Research and Technique Oct 2021Atomic force microscopy (AFM) is a nano-mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction... (Review)
Review
Atomic force microscopy (AFM) is a nano-mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super-resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label-free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM-super-resolution microscopy in biological research.
Topics: Microscopy, Atomic Force; Microscopy, Fluorescence
PubMed: 33955625
DOI: 10.1002/jemt.23800 -
Annual Review of Physical Chemistry Apr 2022Optical microscopy has become an invaluable tool for investigating complex samples. Over the years, many advances to optical microscopes have been made that have allowed... (Review)
Review
Optical microscopy has become an invaluable tool for investigating complex samples. Over the years, many advances to optical microscopes have been made that have allowed us to uncover new insights into the samples studied. Dynamic changes in biological and chemical systems are of utmost importance to study. To probe these samples, multidimensional approaches have been developed to acquire a fuller understanding of the system of interest. These dimensions include the spatial information, such as the three-dimensional coordinates and orientation of the optical probes, and additional chemical and physical properties through combining microscopy with various spectroscopic techniques. In this review, we survey the field of multidimensional microscopy and provide an outlook on the field and challenges that may arise.
Topics: Microscopy
PubMed: 35119943
DOI: 10.1146/annurev-physchem-090519-034100 -
Methods in Cell Biology 2021The recently developed expansion microscopy method (ExM) allows for the resolution of structures below the diffraction limit of light not by sophisticated...
The recently developed expansion microscopy method (ExM) allows for the resolution of structures below the diffraction limit of light not by sophisticated instrumentation, but rather by physically expanding the molecular structure of cells. This happens by crosslinking the protein in the sample to a hydrogel that is polymerized in situ and subsequently expanded, tearing the proteins apart in a nearly isotropic manner. In the resulting, larger facsimile of the original sample, the fluorescence-labeled molecules of interest can be optically separated by conventional fluorescence microscopy since the intermolecular distances are enlarged by a factor ranging from ~4 to 20 depending on the chemistry used for the hydrogel. The achieved improvement in resolution thus corresponds to the expansion factor. Further increase in resolution beyond this value may be achieved by combining ExM with established super-resolution microscopy methods. Indeed, this is possible using structured illumination microscopy (SIM) (Halpern et al., 2017; Wang et al., 2018), single molecule localization microscopy (SMLM) (Zwettler et al., 2020) and stimulated emission depletion (STED), as we and others have shown recently (Gambarotto et al., 2019; Gao et al., 2018; Kim, Kim, Lee, & Shim, 2019; Unnersjö-Jess et al., 2016). Here, we provide a protocol, for our method, called ExSTED, which enabled us to achieve an increase in resolution of up to 30-fold compared to conventional microscopy, well beyond what is possible with conventional STED microscopy. Our protocol includes a strategy to achieve very high intensity fluorescence labeling, which is essential for optimal signal retention during the expansion process for ExSTED.
Topics: Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 33478688
DOI: 10.1016/bs.mcb.2020.06.001 -
Current Protocols Aug 2021Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in... (Review)
Review
Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in 2014. Super-resolution microscopy is increasingly being applied to diverse scientific fields, from single molecules to cell organelles, viruses, bacteria, plants, and animals, especially the mammalian model organism Mus musculus. In this review, we explain how super-resolution microscopy, along with fluorescence microscopy from which it grew, has aided the renaissance of the light microscope. We cover experiment planning and specimen preparation and explain structured illumination microscopy, super-resolution radial fluctuations, stimulated emission depletion microscopy, single-molecule localization microscopy, and super-resolution imaging by pixel reassignment. The final section of this review discusses the strengths and weaknesses of each super-resolution technique and how to choose the best approach for your research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.
Topics: Animals; Biology; Mice; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 34436832
DOI: 10.1002/cpz1.224 -
Nature Reviews. Molecular Cell Biology Jun 2024The proliferation of microscopy methods for live-cell imaging offers many new possibilities for users but can also be challenging to navigate. The prevailing challenge... (Review)
Review
The proliferation of microscopy methods for live-cell imaging offers many new possibilities for users but can also be challenging to navigate. The prevailing challenge in live-cell fluorescence microscopy is capturing intra-cellular dynamics while preserving cell viability. Computational methods can help to address this challenge and are now shifting the boundaries of what is possible to capture in living systems. In this Review, we discuss these computational methods focusing on artificial intelligence-based approaches that can be layered on top of commonly used existing microscopies as well as hybrid methods that integrate computation and microscope hardware. We specifically discuss how computational approaches can improve the signal-to-noise ratio, spatial resolution, temporal resolution and multi-colour capacity of live-cell imaging.
Topics: Humans; Microscopy, Fluorescence; Animals; Image Processing, Computer-Assisted; Artificial Intelligence; Signal-To-Noise Ratio; Cell Survival
PubMed: 38378991
DOI: 10.1038/s41580-024-00702-6 -
Modern Pathology : An Official Journal... Sep 2021Mitoses are often assessed by pathologists to assist the diagnosis of cancer, and to grade malignancy, informing prognosis. Historically, this has been done by... (Review)
Review
Mitoses are often assessed by pathologists to assist the diagnosis of cancer, and to grade malignancy, informing prognosis. Historically, this has been done by expressing the number of mitoses per n high power fields (HPFs), ignoring the fact that microscope fields may differ substantially, even at the same high power (×400) magnification. Despite a requirement to define HPF size in scientific papers, many authors fail to address this issue adequately. The problem is compounded by the switch to digital pathology systems, where ×400 equivalent fields are rectangular and also vary in the area displayed. The potential for error is considerable, and at times this may affect patient care. This is easily solved by the use of standardized international (SI) units. We, therefore, recommend that features such as mitoses are always counted per mm, with an indication of the area to be counted and the method used (usually "hotspot" or "average") to obtain the results.
Topics: Humans; Microscopy; Mitotic Index; Neoplasms
PubMed: 34079071
DOI: 10.1038/s41379-021-00825-7 -
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