Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, which is often hidden behind various sources of noise, convoluted with other information and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review (...) some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem‐specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but also an integral part of the design and implementation of a microscopy experiment. (shrink)

Phototoxicity frequently occurs during live fluorescence microscopy, and its consequences are often underestimated. Damage to cellular macromolecules upon excitation light illumination can impair sample physiology, and even lead to sample death. In this review, we explain how phototoxicity influences live samples, and we highlight that, besides the obvious effects of phototoxicity, there are often subtler consequences of illumination that are imperceptible when only the morphology of samples is examined. Such less apparent manifestations of phototoxicity are equally problematic, and (...) can change the conclusions drawn from an experiment. Thus, limiting phototoxicity is a prerequisite for obtaining reproducible quantitative data on biological processes. We present strategies to reduce phototoxicity, e.g. limiting the illumination to the focal plane and suggest controls for phototoxicity effects. Overall, we argue that phototoxicity needs increased attention from researchers when designing experiments, and when evaluating research findings. (shrink)

Fluorescence microscopy has provided a route to qualitatively analyze features of nuclear structures and chromatin domains with increasing resolution. However, it is becoming increasingly important to develop tools for quantitative analysis. Here, we present an automated method to quantitatively determine the enrichment of several endogenous factors, immunostained in pericentric heterochromatin domains in mouse cells. We show that this method permits an unbiased characterization of changes in the enrichment of several factors with statistical significance from a large number of (...) nuclei. Furthermore, the nuclei can be sorted according to the enrichment value of these factors. This method should prove useful to monitor events related to changes in the amount, rather than the presence or absence, of any factor. By adapting a few parameters, it could be extended to other nuclear structures and the benefit of using available software will permit its use in many biological labs. (shrink)

Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic (...) and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model.Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms Abstract and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis Abstract. (shrink)

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live‐cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the (...) high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. (shrink)

New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for (...) detecting inter‐molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra‐molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio‐temporal protein‐protein interaction studies open new avenues for FRET in biological research. (shrink)

Three‐dimensional structured illumination microscopy (3D‐SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ∼100 nm range. We present first results and assess the potential using 3D‐SIM in combination with 3D fluorescence in situ hybridization (3D‐FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI‐stained DNA in nuclei before (...) and after 3D‐FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship ofXist‐RNA within the inactive X‐territory. These examples demonstrate that an appropriately adapted 3D‐FISH/3D‐SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D‐SIM yields new insights into the functional nuclear organization. (shrink)

Pointillistic based super‐resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super‐resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in (...) photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair‐correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described. (shrink)

Fluorescence microscopy is a powerful tool used in scientific and medical research, but it is inextricably linked to phototoxicity. Neglecting phototoxicity can lead to erroneous or inconclusive results. Recently, several reports have addressed this issue, but it is still underestimated by many researchers, even though it can lead to cell death. Phototoxicity can be reduced by appropriate microscopic techniques and carefully designed experiments. This review focuses on recent strategies to reduce phototoxicity in microscopic imaging of living cells and (...) tissues. We describe digital image processing and new hardware solutions. We point out new modifications of microscopy methods and hope that this review will interest microscopy hardware engineers. Our aim is to underscore the challenges and potential solutions integral to the design of microscopy systems. Simultaneously, we intend to engage biologists, offering insight into the latest technological advancements in imaging that can enhance their understanding and practice. (shrink)

The laser scanning confocal microscope (LSCM)LSCM: laser scanning confocal microscope; FISH: fluorescence in situ hybridisation; DiO6: 3,3′‐dihexyloxacarbocyanine iodide; NBD‐ceramide: 6‐((N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐caproyl)sphingosine; DiO: 3,3′‐dioctadecyloxacarbocyanine perchlorate; DiI: 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate; CCD: charge‐coupled device; DIC: differential interference contrast; FURA2: (‐(2‐(5‐carboxyoxazol‐2‐yl)‐6‐aminobenzofuran‐5‐oxy)‐2‐)2′‐amino‐5′‐methylphenoxy)‐ethane‐N,N,N′,N′‐ tetraacetic acid, sodium salt);BCECF: 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6‐)‐carboxyfluorescein;fluo‐3: 1‐(2‐amino‐5‐(2,7‐dichloro‐6‐hydroxy‐3‐oxo‐3H‐xanthen‐9‐yl)‐2‐(2′amino‐5′‐methylphenoxy)‐ethane‐N,N, N′,N′,‐tetraacetic acid, ammonium salt; DAPI: 4′,6‐diamidino‐2‐phenylindole, dihydrochloride; PET: positron emission tomogrophy; CT: computer‐assisted tomogrophy; CiD: cubitus interruptus dominus; MRC: Medical Research Council; TOTO‐1: benzothiazolium‐4‐quinolinium dimer; YOYO‐1: benzoxazolium‐4‐quinolinium dimer; ex.: excitation wavelength; em.: emission wavelength. is now established as an invaluable tool (...) in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3‐D reconstruction and analysis of light microscope images a practical option. (shrink)

Fluorescence imaging has become an indispensable tool in cell and molecular biology. GFP‐like fluorescent proteins have revolutionized fluorescence microscopy, giving experimenters exquisite control over the localization and specificity of tagged constructs. However, these systems present certain drawbacks and as such, alternative systems based on a fluorogenic interaction between a chromophore and a protein have been developed. While these systems are initially designed as fluorescent labels, they also present new opportunities for the development of novel labeling and detection (...) strategies. This review focuses on new labeling protocols, actuation methods, and biosensors based on fluorogenic protein systems. (shrink)

Conformational changes of proteins and other biomolecules play a fundamental role in their functional mechanism. Single pair Förster resonance energy transfer offers the possibility to detect these conformational changes and dynamics, and to characterize their underlying kinetics. Using spFRET on microscopes with different modes of detection, dynamic timescales ranging from nanoseconds to seconds can be quantified. Confocal microscopy can be used as a means to analyze dynamics in the range of nanoseconds to milliseconds, while total internal reflection fluorescence (...) microscopy offers information about conformational changes on timescales of milliseconds to seconds. While the existence of dynamics can be directly inferred from the FRET efficiency time trace or the correlation of FRET efficiency and fluorescence lifetime, additional computational approaches are required to extract the kinetic rates of these dynamics, a short overview of which is given in this review. The functional mechanism of proteins is often driven by underlying conformational changes. These intramolecular changes and dynamics can be investigated using single pair Förster resonance energy transfer. The kinetic timescales of the dynamics in the range of nanoseconds to seconds is then quantified using different experimental and analytical implementations. (shrink)

Macromolecular interactions play a central role in many biological processes. Protein‐protein interactions have mostly been studied by co‐immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real‐time at the single‐molecule level. This technique is called single‐molecule pull‐down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single‐molecule fluorescence microscopy. SiMPull is used to count (...) how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single‐molecule biochemical tool to perform functional studies on the pulled‐down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools. (shrink)

The purpose of this study is to examine how trends in the use of images in modern life science journals have changed since the spread of computer-based visual and imaging technology. To this end, a new classification system was constructed to analyze how the graphics of a scientific journal have changed over the years. The focus was on one international peer-reviewed journal in life sciences, Cell, which was founded in 1974, whereby 1725 figures and 160 tables from the research articles (...) in Cell were sampled. The unit of classification was defined as a graphic and the figures and tables were divided into 5952 graphics. These graphics were further classified into hierarchical categories, and the data in each category were aggregated every five years. The following categories were observed: data graphics, explanation graphics, and hybrid graphics. Data graphics increased by more than sixfold between 1974 and 2014, and some types of data graphics including mechanical reproduction images and bar charts displayed notable changes. The representation of explanatory graphics changed from hand-painted illustrations to diagrams of Bezier-curves. It is suggested that in addition to the development of experimental technologies such as fluorescent microscopy and big data analysis, continuously evolving application software for image creation and researchers’ motivation to convince reviewers and editors have influenced these changes. (shrink)

Embryos and microscopes share a long, remarkable history and biologists have always been intrigued to watch how embryos develop under the microscope. Here we discuss the advances in microscopy which have greatly influenced our current understanding of embryogenesis. We highlight the evolution of microscopes and the optical technologies that have been instrumental in studying various developmental processes. These imaging modalities provide mechanistic insights into the dynamic cellular and molecular events which drive lineage commitment and morphogenetic changes in the developing (...) embryo. We begin the journey with a brief history of microscopy to study embryos. First, we review the principles and optics of light, fluorescence, confocal, and electron microscopy which have been key techniques for imaging cellular and molecular events during embryonic development. Next, we discuss recent key imaging modalities such as light‐sheet microscopy, which are suitable for whole embryo imaging. Further, we highlight imaging techniques like multiphoton and super resolution microscopy for beyond light diffraction limit, high resolution imaging. Lastly, we review some of the scattering‐based imaging methods and techniques used for imaging human embryos. (shrink)

Six years ago, the Singer lab published a landmark paper which described how individual mRNA particles cross the nuclear pore complex in mammalian tissue culture cells. This involved the simultaneous imaging of mRNAs, each labeled by a large number of tethered fluorescent proteins and fluorescently tagged nuclear pore components. Now two groups have applied this technique to the budding yeast Saccharomyces cerevisiae. Their results indicate that in the course of nuclear export, mRNAs likely engage complexes that are present on either (...) side of the pore and that these interactions are modulated by proteins present in the messenger ribonucleoprotein (mRNP) complex. These findings lend support to the notion that just before and/or after the completion of nuclear export, mRNPs undergo one or more maturation steps that prepare the packaged mRNAs for translation. These results represent new and exciting insights into the mechanism of mRNA nuclear export. (shrink)

Herculaneum papyri found during the discovery of the Villa dei Papiri in the XVIII century are our only knowledge about Greek philosophical schools. Unfortunately, the original manuscripts are in a precarious state of conservation and the currently available editions of them have largely been made obsolete by the latest technological progress. The aim of the Advanced Grant ERC project ‘Greekschools’ is to provide a new protocol based on optical methods to increase the text reading and thus allow for a new (...) critical edition of the whole treatise of Philodemus’ "Arrangement of the Philosophers". A multi-disciplinary approach is adopted and non-invasive techniques to Herculaneum papyri investigations such as Macro X-Ray Fluorescence Imaging (MA-XRF), Shortwave-Infrared Hyperspectral Imaging (SWIR), high resolution digital microscopy and Technical Photography were applied to read the text hidden on the verso, detect, classify, and replace overlapping layers and read the text concealed inside them. In this work some first preliminary results will be reported. (shrink)

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how (...) the impact of modern technologies is influenced not only by further hard‐ and software developments, but also by the availability and suitability of protein‐engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. (shrink)

Light sheet microscopy is an easy to implement and extremely powerful alternative to established fluorescence imaging techniques such as laser scanning confocal, multi‐photon and spinning disk microscopy. By illuminating the sample only with a thin slice of light, photo‐bleaching is reduced to a minimum, making light sheet microscopy ideal for non‐destructive imaging of fragile samples over extended periods of time. Millimeter‐sized samples can be imaged rapidly with high resolution and high depth penetration. A large variety of (...) instruments have been developed and optimized for a number of different samples: Bessel beams form thin light sheets for single cells, and selective plane illumination microscopy (SPIM) offers multi‐view acquisition to image entire embryos with isotropic resolution. This review explains how light sheet microscopy involves a conceptually new microscope design and how it changes modern imaging in biology. (shrink)

The examination of the complex cell biology of the human malaria parasite Plasmodium falciparum usually relies on the time‐consuming generation of transgenic parasites. Here, metabolic labeling and click chemistry are employed as a fast transfection‐independent method for the microscopic examination of protein S‐palmitoylation, an important post‐translational modification during the asexual intraerythrocytic replication of P. falciparum. Applying various microscopy approaches such as confocal, single‐molecule switching, and electron microscopy, differences in the extent of labeling within the different asexual developmental stages (...) of P. falciparum and the host erythrocytes over time are observed. (shrink)

Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination – converting two photons into one photon of half the incident wavelength – and can be detected by conventional two‐photon microscopy. Because the optical signal of SHG nanoprobes is based (...) on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long‐term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution. (shrink)

Gap junction intercellular communication channels permit the exchange of small regulatory molecules and ions between neighbouring cells and coordinate cellular activity in diverse tissue and organ systems. These channels have short half‐lives and complex assembly and degradation pathways. Much of the recent work elucidating gap junction biogenesis has featured the use of connexins (Cx), the constituent proteins of gap junctions, tagged with reporter proteins such as Green Fluorescent Protein (GFP) and has illuminated the dynamics of channel assembly in live cells (...) by high‐resolution time‐lapse microscopy. With some studies, however, there are potential short‐comings associated with the GFP chimeric protein technologies. A recent report by Gaietta et al., has highlighted the use of recombinant proteins with tetracysteine tags attached to the carboxyl terminus of Cx43, which differentially labels ‘old’ and ‘new’ connexins thus opening up new avenues for studying temporal and spatial localisation of proteins and in situ trafficking events.1 BioEssays 24:876–880, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of (...) class='Hi'>fluorescence fluctuation spectroscopy. (shrink)

Although negative stain electron microscopy is a wonderfully simple way of directly visualizing protein complexes and other biological macromolecules, the images are not really comparable to those of objects seen in everyday life. The failure to appreciate this has recently led to an incorrect interpretation of RecA‐family filament structures.

Emergence of the novel pathogenic coronavirus SARS‐CoV‐2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi‐quantitative high‐content microscopy‐based assay for detection of three major classes (IgG, IgA, and IgM) of SARS‐CoV‐2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi‐automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS‐CoV‐2 serological assays. Sensitive, (...) specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high‐throughput microscopy to rapidly develop serological assays for emerging virus infections. (shrink)

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution‐based single molecule technique, fluorescence correlation spectroscopy (FCS). This time‐averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven (...) to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual‐color cross‐correlation and two‐photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research. BioEssays 24:758–764, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

In Amerindian ontologies, hallucinations or visions, rather than being dismissed as delusions or symbolic constructs, are recognized as means of perceptual access to physical reality. Lowland South American shamans claim to be able to diagnose and treat infectious diseases, and to assess the status of wildlife resources through interactions with pathogenic agents perceived in visions. This essay examines some perceptual capabilities that shamans might be employing to explore their physical reality. The structure of the eye affords a form of (...) class='Hi'>microscopy of retinal structures and of objects flowing within them, including cells and microbial agents during systemic infection. Lowland South American shamanic practices involve optical and physiological conditions that optimize entoptic microscopy. Images of those visions display the characteristic features of shadow formation, confirming their microscopic origin. This phenomenological access to the microscopic world and similarities with the panorama depicted by current microbiology indicate the commensurability of these forms of knowledge. (shrink)

This paper deals with an analysis of debris produced during the polishing of diamond. The debris is carefully collected 'as ejected' to shorten the history of the freshly removed material. Using high-resolution electron microscopy as well as electron-energy-loss spectroscopy, the structure of the material is revealed and analysed in terms of density, percentage of sp 2 hybridized carbon, and oxygen content. Debris from polishing in the so-called hard and soft directions were involved in this investigation. Overall the structure of (...) all debris is amorphous carbon. The material appears to be composed of small clusters, some nanometres in diameter, in which the graphite basal planes can be recognized. Very few and very small nanometre-sized diamond particles were found in the debris from polishing in the hard direction. The results support a polishing mechanism based on a mechanically induced transformation of diamond to graphite, after which material removal easily occurs. The well-known anisotropy observed in polishing can be explained satisfactorily on the basis of this model. Finally, in appendices, the art of polishing and the role of the black powder during preparation of the scaife are discussed. (shrink)

A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. (...) Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180–187, 2000. ©2000 John Wiley & Sons, Inc. (shrink)

Cryo‐electron microscopy allows the visualization of macromolecules in their native state. Combined with techniques of three‐dimensional reconstruction, cryo‐EM images of single molecules can be used to study macromolecular interactions. The ribosome, a large RNA–protein complex with multiple binding interactions, is an excellent test case illustrating the power of these new techniques. Conformational changes during the binding of tRNA and protein factors to the ribosome can now be studied without the interference of crystal packing. Now that the first X‐ray structures (...) of ribosomal subunits have become available, conformational changes observed by cryo‐EM in different functional states can be traced back to internal rearrangements of the underlying structural framework. Electron microscopy, X‐ray crystallography, and modeling should be used together in the endeavor to understand the functioning of the translational machinery. BioEssays 23:725–732, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

Microscopy allows us to observe objects we cannot see with our eyes alone. With a light microscope, we can distinguish objects at the scale of the wavelengths of visible light just under a micrometer. Around 1870 Ernst Abbe, who laid the foundation of modern optics, suggested that the resolution of a microscope would improve by using some yet-unknown radiation with shorter wavelengths than visible light, that is, below 390 nanometers (1 nm = 10−9 m). Electrons can have wavelengths near (...) 1 picometer (1 pm = 10−12 m) and should therefore allow atoms to be distinguished since they are typically at least a few hundred pm apart. In this oversimplified view, further decreasing the wavelength of the radiation should allow us to increase the resolution of electron microscopy even more. However, this can only be done by increasing the energy of the radiation, which would ultimately destroy the samples. Other factors also affect the resolution of electron microscopes, among them non-ideal imaging properties of electromagnetic lenses, which result in false images. It took more than half a century to understand and control these aberrations and separate objects less than 1 Ångstrom (1 Å = 10−10 m) apart. (shrink)

An in-situ transmission electron microscopy straining technique has been used to investigate the dynamics of dislocation-defect interactions in ion-irradiated copper and the subsequent formation of defect-free channels. Defect removal frequently required interaction with multiple dislocations, although screw dislocations were more efficient at annihilating defects than edge dislocations were. The defect pinning strength was determined from the dislocation curvature prior to breakaway and exhibited values ranging from 15 to 175 MPa. Pre-existing dislocations percolated through the defect field but did not (...) show long-range motion, indicating that they are not responsible for creating the defect-free channels and have a limited contribution to the total plasticity. Defect-free channels were associated with the movement of many dislocations, which originated from grain boundaries or regions of high stress concentration such as at a crack tip. These experimental results are compared with atomistic simulations of the interaction of partial dislocations with defects in copper and a dispersed-barrier-hardening crystal plasticity model to correlate the observations to bulk mechanical properties. (shrink)

The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an instrument that can access all scales relevant to modern biology – from individual molecules all the way to whole tissues and organisms and from single snapshots to time‐lapse recordings sampling from milliseconds to days. As these and more new technologies appear, the challenges of delivering them to the community grows as well. I discuss some of (...) these challenges, and the examples where openly shared technology have made an impact on the field. (shrink)