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.

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)

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)

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)

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)

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)

SummaryThis paper analyses the development of three methods for detecting bloodstains during the first half of the nineteenth-century in France. After dealing with the main problems in detecting bloodstains, the paper describes the chemical tests introduced in the mid-1820s. Then the first uses of the microscope in the detection of bloodstains around 1827 are discussed. The most controversial method is then examined, the smell test introduced by Jean-Pierre Barruel in 1829, and the debates which took place in French academies and (...) learned societies during ensuing years are surveyed. Moving to the courtrooms a review is conducted of how the different methods were employed in criminal trials. By reviewing these cases, the main arguments against Barruel's test during the 1830s are explored as well as the changes making possible the return of the microscope to legal medicine around 1840. By reconstructing the history of these three methods, the paper reveals how the senses of smell and vision were employed in order to produce convincing evidence in both academies and courts. The paper questions two linear master narratives that are organized in terms of progress and decline: the development of forensic science as a result of continued technological progress; and the supposed decline of smell in the history of the senses, particularly in the realm of chemistry and medicine. (shrink)

In very recent Leibniz commentary, there has been a movement among some commentators toward the view that Leibniz was not an unwavering monadological immaterialist, committed to the substantiality only of absolutely simple, immaterial nodes of perception and appetite. It has been conceded that Leibniz was also partially sympathetic to an ontology that would concede full substantiality to composite entities. Most commentators who have been willing to concede as much have confined this alternative metaphysics to Leibniz's middle period, roughly 1676--1694. These (...) commentators, moreover, have remained fairly silent as to the reasons why Leibniz might wish to consider an ontology at such odds with his well-known immaterialism. In this dissertation, I contribute to the discussion of Leibniz's composite-substance metaphysics in three ways. First of all, I am going to argue that we should see the composite-substance metaphysics as an alternative Leibniz sustained until the very end of his career. Second of all, I provide an account of Leibniz's motivation for sustaining this alternative metaphysics, arguing that the composite-substance metaphysics is first and foremost the result of Leibniz's effort to provide a philosophical interpretation of the recent discovery of the apparent ubiquity of microscopic life. Finally, I vindicate a number of 18th-century, microorganic and physicalistic interpretations of Leibniz's theory of monads, particularly those of the Francophone natural scientists, against the view among 20th-century commentators that Leibniz was grossly misinterpreted in the decades following his death in 1716. I show that 18th-century, thinkers didn't so much misinterpret Leibniz, as they did emphasize one, currently very unfamiliar, tendency in Leibniz's thought on the nature of substance. (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)

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)

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)

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)

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)

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)

The ArgumentContrary to the dominant historiography of microscopy, which tends to maintain that there was no microscopical program in the Enlightenment, this paper argues that there was such a program and attempts to illustrate one aspect of its dynamic character. The experiments, observations, and interpretations on rotifers and their management by scholars of that period show that there did exist a precise axis of research that can be followed historically. Indeed, the various controversies these scholars engaged in imply that (...) they performed accurate microscopical experiments and observations and also carefully interpreted their visual data. Furthermore, the kinds of phenomena presented by the rotifer and other microscopic entities, such as their morphology or their revival from desiccation, helped to improve methodology on the use of the microscope. The paper also argues that this sort of inquiry is not fundamentally different from other research in the natural sciences of the eighteenth century. (shrink)

Supersaturated hcp f -Zr alloys containing 22-28 at.% N were prepared by nitriding sheets of Zr in an atmosphere of high-purity N 2 , followed by homogenization under high-purity Ar gas. Quenching and isothermal ageing of the alloys for various times between 500 and 650°C resulted in precipitation of a metastable phase, rather than the equilibrium phase ZrN. This investigation focused on determining the structure, orientation relationship, habit plane, morphology, growth kinetics and atomic growth mechanism of this non-equilibrium precipitate using (...) transmission electron microscopy imaging and diffraction techniques, high-resolution transmission electron microscopy, electron-energy-loss spectroscopy and various simulation programs. The precipitate, which was arbitrarily designated the  phase, has a monoclinic Bravais lattice. Its lattice parameters are a = 0.32 nm, b = 0.60 nm, c = 0.56 nm, f = g = 90° and n = 121.5°. Its orientation relationship with the f matrix is $$ _alpha /mskip-2/ _xiquad adquad [01bar 10] _alpha /mskip-2/ [001]_xi, $$ and the average habit plane of lenticularly shaped precipitates is $_alpha$ . Determination of the structure and other aspects of the phase transformation are discussed. (shrink)