-
Nature Methods Jan 2019Many biological investigations require 3D imaging of cells or tissues with nanoscale spatial resolution. We recently discovered that preserved biological specimens can... (Review)
Review
Many biological investigations require 3D imaging of cells or tissues with nanoscale spatial resolution. We recently discovered that preserved biological specimens can be physically expanded in an isotropic fashion through a chemical process. Expansion microscopy (ExM) allows nanoscale imaging of biological specimens with conventional microscopes, decrowds biomolecules in support of signal amplification and multiplexed readout chemistries, and makes specimens transparent. We review the principles of how ExM works, advances in the technology made by our group and others, and its applications throughout biology and medicine.
Topics: Biomedical Research; Microscopy
PubMed: 30573813
DOI: 10.1038/s41592-018-0219-4 -
Annual Review of Biophysics May 2023Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution... (Review)
Review
Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.
Topics: Single Molecule Imaging; Microscopy, Fluorescence
PubMed: 37159293
DOI: 10.1146/annurev-biophys-111622-091212 -
BMC Biology Sep 2018Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in...
Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.
Topics: Cytological Techniques; Electron Microscope Tomography; Imaging, Three-Dimensional; Microscopy, Electron, Scanning; Microscopy, Fluorescence
PubMed: 30189863
DOI: 10.1186/s12915-018-0560-1 -
Trends in Biotechnology Feb 2020The spatial distribution of molecules and cells is fundamental to understanding biological systems. Traditionally, microscopies based on electromagnetic waves such as... (Review)
Review
The spatial distribution of molecules and cells is fundamental to understanding biological systems. Traditionally, microscopies based on electromagnetic waves such as visible light have been used to localize cellular components by direct visualization. However, these techniques suffer from limitations of transmissibility and throughput. Complementary to optical approaches, biochemical techniques such as crosslinking can colocalize molecules without suffering the same limitations. However, biochemical approaches are often unable to combine individual colocalizations into a map across entire cells or tissues. Microscopy-by-sequencing techniques aim to biochemically colocalize DNA-barcoded molecules and, by tracking their thus unique identities, reconcile all colocalizations into a global spatial map. Here, we review this new field and discuss its enormous potential to answer a broad spectrum of questions.
Topics: DNA Barcoding, Taxonomic; High-Throughput Nucleotide Sequencing; Microscopy; Molecular Imaging
PubMed: 31416630
DOI: 10.1016/j.tibtech.2019.06.001 -
Microscopy (Oxford, England) Jun 2023Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely...
Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.
Topics: Humans; Virus Diseases; Viruses; Microscopy, Atomic Force; Microscopy, Electron; Microscopy, Fluorescence; Cryoelectron Microscopy
PubMed: 37079744
DOI: 10.1093/jmicro/dfad024 -
Plant Physiology Apr 2020Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plant molecular biology, agronomy, plant... (Review)
Review
Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plant molecular biology, agronomy, plant physiology, plant nutrition, and ionomics. However, it is often challenging to evaluate the applicability of the wide range of techniques available, with each having its own strengths and limitations. Here, we compare scanning/transmission electron microscopy-based energy-dispersive x-ray spectroscopy, x-ray fluorescence microscopy, particle-induced x-ray emission, laser ablation inductively coupled plasma-mass spectrometry, nanoscale secondary ion mass spectroscopy, autoradiography, and confocal microscopy with fluorophores. For these various techniques, we compare their accessibility, their ability to analyze hydrated tissues (without sample preparation) and suitability for in vivo analyses, as well as examining their most important analytical merits, such as resolution, sensitivity, depth of analysis, and the range of elements that can be analyzed. We hope that this information will assist other researchers to select, access, and evaluate the approach that is most useful in their particular research program or application.
Topics: Mass Spectrometry; Microscopy, Confocal; Microscopy, Electron; Plants; Spectrometry, X-Ray Emission
PubMed: 31974126
DOI: 10.1104/pp.19.01306 -
Current Protocols in Cytometry Oct 2014Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with... (Comparative Study)
Comparative Study Review
Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation--integration.
Topics: Animals; Humans; Microscopy, Electron; Microscopy, Fluorescence
PubMed: 25271959
DOI: 10.1002/0471142956.cy1236s70 -
The International Journal of... 2019In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring... (Review)
Review
In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring during oogenesis and early embryonic development in mammals. In short, two modes of cellular imaging are now available: those involving fluorescent labels and those which are fluorophore-free. Fluorescence microscopy, in its various forms, is used predominantly in research, as it provides detailed information about cellular processes; however, it can involove an increased risk of photodamage. Fluorophore-free techniques provide, on the other hand, a smaller amount of biological data but they are safer for cells and therefore can be potentially used in a clinical setting. Here, we review various fluorescence and fluorophore-free visualisation approaches and discuss their applicability in developmental biology and reproductive medicine.
Topics: Blastocyst; Embryonic Development; Microscopy; Microscopy, Fluorescence; Microscopy, Polarization; Oocytes
PubMed: 31058300
DOI: 10.1387/ijdb.180300aa -
Current Opinion in Microbiology Jun 2018Understanding how microbes utilize their environment is aided by visualizing them in their natural context at high resolution. Correlative imaging enables efficient... (Review)
Review
Understanding how microbes utilize their environment is aided by visualizing them in their natural context at high resolution. Correlative imaging enables efficient targeting and identification of labelled viral and bacterial components by light microscopy combined with high resolution imaging by electron microscopy. Advances in genetic and bioorthogonal labelling, improved workflows for targeting and image correlation, and large-scale data collection are increasing the applicability of correlative imaging methods. Furthermore, developments in mass spectroscopy and soft X-ray imaging are expanding the correlative imaging modalities available. Investigating the structure and organization of microbes within their host by combined imaging methods provides important insights into mechanisms of infection and disease which cannot be obtained by other techniques.
Topics: Bacteria; Communicable Diseases; Host Microbial Interactions; Humans; Mass Spectrometry; Microscopy; Microscopy, Electron; Single Molecule Imaging; Viruses
PubMed: 29414444
DOI: 10.1016/j.mib.2018.01.009 -
Current Opinion in Structural Biology Aug 2023Super-resolution microscopy is a series of imaging techniques that bypass the diffraction limit of resolution. Since the 1990s, optical approaches, such as... (Review)
Review
Super-resolution microscopy is a series of imaging techniques that bypass the diffraction limit of resolution. Since the 1990s, optical approaches, such as single-molecular localization microscopy, have allowed us to visualize biological samples from the sub-organelle to the molecular level. Recently, a chemical approach called expansion microscopy emerged as a new trend in super-resolution microscopy. It physically enlarges cells and tissues, which leads to an increase in the effective resolution of any microscope by the length expansion factor. Compared with optical approaches, expansion microscopy has a lower cost and higher imaging depth but requires a more complex procedure. The integration of expansion microscopy and advanced microscopes significantly pushed forward the boundary of super-resolution microscopy. This review covers the current state of the art in expansion microscopy, including the latest methods and their applications, as well as challenges and opportunities for future research.
Topics: Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 37253290
DOI: 10.1016/j.sbi.2023.102614