1,720,966 research outputs found
NANOSCALE INVESTIGATION OF NUCLEAR STRUCTURES BY TIME-RESOLVED MICROSCOPY
No full textThe eukaryotic cell nucleus is composed by heterogeneous biological structures, such as the nuclear envelope (NE) and chromatin. At a morphological level, chromatin organization and its interactions with nuclear structures, such as nuclear lamina (NL) and nuclear pore complex (NPC), are suggested to play an essential role in the regulation of gene activity, which involves the packaging of the genome into transcriptionally active and inactive sites, bound to healthy cell proliferation and maintenance. However, the processes governing the relation between nuclear structures and gene regulation are still unclear. For this reason, the advanced microscopy methods represent a powerful tool for imaging nuclear structures at the nanometer level, which is essential to understand the effect of nuclear interactions on genome function. The nanometer information may be achieved either through the advanced imaging techniques in combination with fluorescence spectroscopy or with the help of super-resolution methods, increasing the spatial resolution of the conventional optical microscopy. In this thesis, I implemented a double strategy based on a novel FLIM-FRET assay and super resolution SPLIT-STED method for the investigation of the chromatin organization and nuclear envelope components (lamins and NPC) at the nanoscale, in combination with the phasor analysis. The phasor approach can be applied to several fluorescence microscopy techniques abled to provide an image with an additional information in a third channel. Phasor plot is a graphical representation, which decodes the fluorescence dynamics encoded in the image, revealing a powerful tool for the data analysis in time-resolved imaging.
The Chapter 1 of the thesis is characterized by an Introduction, which provides an overview on the chromatin organization at the nanoscale and the description of the several advanced fluorescence microscopy techniques used for its investigation. They are broadly divided into two main categories: the advanced imaging techniques, such as Fluorescence Correlation Spectroscopy (FCS), single particle tracking (SPT) and Fluorescence Recovery After Photobleaching (FRAP), Forster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) and the super-resolution techniques, which include Stimulated Emission Depletion (STED), Structured Illumination Microscopy (SIM) and single molecule localization microscopy (SMLM). Following, Chapter 2 focus on the capabilities of the phasor approach in time-resolved microscopy, as a powerful tool for the analysis of the experimental data. After a description of the principles of time-domain and frequency-domain measurements, in this section are explained the rules of the phasor analysis and its applications in different fluorescence microscopy techniques. In Chapter 3, I present a FRET assay, based on the staining of the nuclei with two DNA-binding dyes (e.g. Hoechst 33342 and Syto Green 13) by using frequency-domain detection of FLIM and the phasor analysis in live interphase nuclei. I show that the FRET level strongly depends on the relative concentration of the two fluorophores. I describe a method to correct the values of FRET efficiency and demonstrate that, with this correction, the FLIM-FRET assay can be used to quantify variations of nanoscale chromatin compaction in live cells. In Chapter 4, the phasor analysis is employed to the improvement of the resolving power of the super-resolution STED microscopy. I describe a novel method to investigate nuclear structures at the nanometer level, known as SPLIT (Separation of Photons by Lifetime Tuning), developed by my group in last years. By using the phasor approach, the SPLIT technique decodes the variations of spectroscopic parameters of fluorophores, such as lifetime and fluorescence intensity, due to the effect of the modulated depletion power of the STED technique, increasing the resolving power. In this chapter, I develop the concept of the SPLIT method modulating the excitation pattern during the image acquisition to overcome its limitation linked to the photobleaching effect and the signal-to-noise ratio
Chromatin Nanoscale Organization Investigated by FLIM-FRET and STED Superresolution Microscopy
No full textA Novel Viewpoint to Analyze Structured Illumination Microscopy (Sim) Data
No full textStructured Illumination Microscopy (SIM) is an important tool among the Super Resolution Microscopy techniques [1]. SIM stands out for its capability to perform optical sectioning, multicolor channel acquisition and live cell imaging, thanks to its low phototoxicity. In SIM, the specimen is illuminated with a high-frequency grating pattern, instead of a uniform field. The multiplication of sample features and this pattern gives rise to Moiré fringes.The obtained interference pattern allows encoding of sub-diffraction sample's features into lower frequencies collected by the objective lens. The reconstruction process then decodes otherwise unresolvable high-frequency information creating the super-resolved image.Here we aim to analyze SIM data from a rather different standpoint, based on the idea of Separation of Photons by LIfetime Tuning (SPLIT)[2]. In SPLIT the sub-diffraction spatial information is encoded into an additional channel of the microscope. The information encoded within this channel is visualized and analyzed in the phasor plot and allows separating a fractional image component of higher spatial resolution corresponding to the center of the PSF.In the context of Stimulated Emission Depletion (STED) microscopy, we recently demonstrated that SPLIT can be used to decode spatial information encoded in the nanosecond fluorescence lifetime [2] or variations of the depletion power [3]. We show here that in the case of SIM it is possible to use SPLIT to analyze the information encoded into the images acquired at different illumination patterns. In particular an important aspect of SPLIT is that the Phasor Plot provides a visual, intuitive and direct evaluation of the acquired data [3].We demonstrate that knowledge of the illumination pattern can be used to perform SPLIT analysis and to reconstruct a super-resolved image bypassing the traditional Fourier reconstruction.As an application, we use the SPLIT-SIM approach to perform super-resolution imaging of chromatin-related structures
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
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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