1,721,032 research outputs found
The nucleus under the microscope. A biophysical approach.
No full textAt the beginning of 1950, many researchers challenged the possibility to overcome the fundamental Abbe limit. An attempt was made by Giuliano Toraldo di Francia, who showed that the width of the point-spread function can be reduced applying a filtering technique (called apodozation) (1). In 1994, a revolutionary event took place in the field of optical microscopy: Hell described a method for circumventing the light diffraction barrier (2). In this way, details that were not visible in diffraction-limited techniques could be imaged using a fluorescence microscopy. Nowadays, these methods termed Far-field fluorescence microscopy or nanoscopy techniques, has become an indispensable tool for scientist to address important biological and biophysical questions at the single molecule level. To highlight the outstanding importance of such techniques, the Royal Swedish Academy of Sciences awarded Eric Betzig, Stefan W. Hell, and William E. Moerner the Nobel Prize in Chemistry 2014 “for the development of super-resolved fluorescence microscopy”. In addition, several important technical improvements, including confocal laser scanning microscopy (CLSM) (3), multiphoton microscopy, 4Pi (4) and I5M (5) have had an important role in the field of optical microscopy.
On the other side, in 2015 Boyden and colleagues developed a new method termed Expansion Microscopy (ExM), which allows expanding uniformly biological samples by increasing the relative distances among fluorescent molecules labelling specific cellular components (6). ExM permits to achieve a lateral resolution of about 65 nm, using a conventional - diffracted microscope. However, all super resolution methods demand a particular attention in the sample preparation. Achieving super resolved images require the optimization of every steps involved in the labelling process, from the expression of a fluorescent proteins to the fixation of the biological samples. In the last years, these labelling strategies have obtained a critical role in the field of fluorescence microscopy. In particular, the design and the localization precision of specific affinity probes are crucial features that can restrict the applicability of these techniques. In this work, several labelling approaches and optimization of different staining protocol for super resolution techniques were addressed. My effort was focused on STED nanoscopy and ExM, and how to optimize the labelling protocol, the fluorophores choice for a high labelling density. The optimization of the steps involved in the labelling processes allows me combining ExM with STED nanoscopy (ExSTED), to enhance the final resolution (7). In addition, these techniques were used to decipher molecular assemblies in the cellular nuclei. In particular, my attention was focused on an important layer termed nuclear envelope (NE) (8).
This nuclear region encases the genetic material, maintains the regular shape of the nucleus
and regulates the gene expression. NE is composed by two lipid bilayer and different class of
proteins, which pass through or are strictly linked to the nuclear membranes. Nuclear pore
complexes (NPCs) and nuclear lamins, two classes of proteins belonging to the NE, were
investigated in this work. In particular, NPCs was used to evaluate the isotropy and calculate
the expansion factor (EF) at the nanoscale level in ExM. In this work, we show that Nup153, a
filamentous subunit localized in the nuclear pore basket (9), is a good reporter to verify the
isotropy of the expansion process and its quantification. In addition, nuclear lamins, in
particular lamin A (LA) and its mutation ΔLA50 (10), were used to investigate the physiological
and pathological nuclear membrane invagination in normal and aging cells
Expansion and Light‐Sheet Microscopy for Nanoscale 3D Imaging
Full text linkExpansion Microscopy (ExM) and Light-Sheet Fluorescence Microscopy (LSFM) are forefront imaging techniques that enable high-resolution visualization of biological specimens. ExM enhances nanoscale investigation using conventional fluorescence microscopes, while LSFM offers rapid, minimally invasive imaging over large volumes. This review explores the joint advancements of ExM and LSFM, focusing on the excellent performance of the integrated modality obtained from the combination of the two, which is refer to as ExLSFM. In doing so, the chemical processes required for ExM, the tailored optical setup of LSFM for examining expanded samples, and the adjustments in sample preparation for accurate data collection are emphasized. It is delve into various specimen types studied using this integrated method and assess its potential for future applications. The goal of this literature review is to enrich the comprehension of ExM and LSFM, encouraging their wider use and ongoing development, looking forward to the upcoming challenges, and anticipating innovations in these imaging techniques
The Nucluear Pore Complex as Intrinsic Reporter for Isotropic Expansion Microscopy
No full textExpansion microscopy (ExM) is a super-resolution imaging method that does not require any special optical microscope(1). The key point resides on the possibility of uniformly chemically expanding a sample, thus increasing the relative distances among objects of interest as fluorescent molecules labeling specific components. ExM is highly invasive; it involves gelation and digestion steps that could introduce artifacts and heterogeneities in the relative spatial distribution of complex proteins in the cells. The possibility to combine STED(2) and ExM (ExSTED)(3) allows not only an unprecedented resolution but also a higher sensitivity to discover possible pitfalls. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters, i.e., scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC)(4). In particular, we show that Nup153, a filamentous subunit localized in the nuclear pore basket(5), is an excellent reporter to address the isotropy of the expansion process quantitatively. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale with consistent accuracy in the range of 20 nm. It is an excellent method for structural studies of macromolecular complexe
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Pioglitazone Phases and Metabolic Effects in Nanoparticle-Treated Cells Analyzed via Rapid Visualization of FLIM Images
Full text linkFluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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