Fish embryo multimodal imaging by laser Doppler digital holography

A laser Doppler imaging scheme combined to an upright microscope is proposed. Quantitative Doppler imaging in both velocity norm and direction, as well as amplitude contrast of either zebrafish flesh or vasculature is demonstrated.Comment: Signal Recovery and Synthesis, Jul 2014, Seattle, United States. (Optical Society of America), OSA Technical Digest (online) 2014, paper JTu4A.7., Imaging and Applied Optics 2014, http://www.opticsinfobase.org/abstract.cfm?URI=SRS-2014-JTu4A.

Spliceosomal Prp24 Unwinds A Minimal U2/u6 Complex From Yeast

Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA to form mature mRNA. Pre-mRNAs undergo splicing to remove introns, non–protein coding regions, and religate exons, protein coding regions. This process is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and numerous protein factors. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal. Several spliceosomal proteins facilitate structural rearrangements important for spliceosomal assembly and function. Prp24 is an essential factor in U6 snRNP assembly, and it has been proposed to assist in U4/U6 formation and unwinding. Here, we address the question whether Prp24 affects the U2/U6 complex dynamics. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we have previously shown that a minimal U2/U6 complex from yeast can adopt at least three distinct conformations in dynamic equilibrium. Our new single molecule data show that Prp24 unwinds U2 from U2/U6 complex and stabilizes U6 in a low FRET conformation. We also show that the RNA Recognition Motifs of Prp24 affect the binding affinity of Prp24 for U6 and unwinding activity. We propose that Prp24 plays an important role in U2 and U6 snRNP recycling by dissociating the U2/U6 complex

Watching dynamics and assembly of spliceosomal complexes at single molecule resolution

Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA to form mature mRNA. Splicing is catalysed by the spliceosome; a ribonucleoprotein complex consisting of five small nuclear ribonucleoproteins (snRNPs) and over 100 proteins. Proper assembly of spliceosomal components is critical for its function, and thus assembly defects can be lethal. Several diseases have been associated with splicing defects, such as cancer (breast cancer, leukaemia), cystic fibrosis, duchenne muscular dystrophy (DMD), retinitis pigmentosa (RP) and taybi-linder syndrome (TALS). Studying the structural dynamics and distinct functions of snRNA complexes and the factors that affect the stability of those complexes provides an overall idea regarding the structure and function of the spliceosome, which can guide us to discover novel therapeutics for splicing related diseases. Hence, the aim of this study is employing smFRET technique to monitor the structural dynamics and assembly of snRNA complexes and the effect of protein factors on those dynamics with single molecule resolution. Three specific aims have been addressed in this thesis work to achieve the main goal. The first part of this study is focused on understanding how spliceosomal components are recycled. This work shows that binding of Prp24; U6 snRNP specific protein unwinds U2 from U2/U6 complex and stabilizes U6 at a low FRET conformation, suggesting a novel role for Prp24 as a recycling factor. The second part of this study is focused on understanding the assembly of sub-spliceosomal complexes and their global structure. This work shows that although the binding of individual proteins slightly changes the conformation of U4/U6 duplex; overall it maintains a rigid structure. This suggests that the U4/U6 adopts a preformed conformation and act as a scaffold for protein binding, while preventing U6 from premature activation. The third part of this study is focused on understanding structural and functional similarities between minor and major spliceosomal complexes. This work shows that minor spliceosomal U12/U6atac complex adopts a conformation similar to the three-helix junction structure of major spliceosomal U2/U6.Open Acces

VANGUARD: A Blockchain-Based Solution to Digital Piracy

Online pirates and Intellectual Property (IP) holders have been in perpetual war over various products like music, movies, software, etc. since the popularity of the Internet. It is estimated that the US entertainment industry loses approximately 29 billion USD every year for pirates. Online piracy has since gone from bad to worse as growing internet users and better broadband connections enable people to share large files freely over the internet. The objective of this research is to investigate the causes and enablers for online piracy in movie industry and to come up with an anti-pirating solution. The primary outcome of the study will consist of a dedicated blockchain based anti-piracy system, 2018;Vanguard2019;. This system will provide all-round piracy protection from a built-in streaming service to a component to actively look through the internet for pirated movies and torrents. This system will greatly deter the piracy of movies since the IP holders can ensure their IP rights through this system and quickly act against illegitimate distribution of their media

Imaging Gold Nanoparticles in Living Cells Environments using Heterodyne Digital Holographic Microscopy

This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environment. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total internal reflection configuration with holographic microscopy. Due to a higher scattering efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of a gold marker is obtained within a 3D mapping of the entire sample's scattered field, with a lateral precision of 5 nm and 100 nm in the x,y and in the z directions respectively, demonstrating the ability of holographic microscopy to locate nanoparticles in living cells environments

Dark-field digital holographic microscopy for 3D-tracking of gold nanoparticles

We present a new technique that combines off-axis Digital Holography and Dark Field Microscopy to track 100nm gold particles diffusing in water. We show that a single hologram is sufficient to localize several particles in a thick sample with a localization accuracy independent of the particle position. From our measurements we reconstruct the trajectories of the particles and derive their 3D diffusion coefficient. Our results pave the way for quantitative studies of the motion of single nanoparticle in complex media

Quantitative Phase Imaging Microscopy with Multi-Wavelength Optical Phase Unwrapping

This dissertation presents a quantitative phase imaging microscopy technique that combines phase-shifting interferometry with multi-wavelength optical phase unwrapping. The technique consists of a Michelson-type interferometer illuminated with any of three types of light sources; light emitting diodes, laser diodes and a ring dye laser. Interference images are obtained by using a 4-frame phase shifting method, and are combined to calculate the phase of the object surface. The 2π ambiguities are removed by repeating the experiment combining two and three different wavelengths, which yields phase images of effective wavelength much longer than the original. The resulting image is a profile of the object surface with a height resolution of several nanometers and range of several microns. To our knowledge, this is the first time that a three wavelength optical phase unwrapping method with no amplified phase noise has been presented for fullframe phase images. The results presented here are divided into three main categories based on the source of illumination; light emitting diodes, laser diodes and a ring dye laser. Results for both two-wavelength optical unwrapping and three-wavelength optical unwrapping techniques are demonstrated. The interferographic images using broadband sources such as light emitting diodes are significantly less affected by coherent noise compared to images obtained using lasers. Our results show that the three wavelength optical phase unwrapping can also be effectively applied to unwrap phase images obtained using coherent light sources such as lasers and laser diodes, without amplifying phase noise in the final phase image. We have successfully shown that our multi-wavelength phase-shifting technique extends the range free of 2π ambiguities in the phase map without using conventional computation intensive phase unwrapping methods. This phase imaging technique can be used to measure physical thickness or height of both biological and other microscopic samples, with nanometer axial resolution. An added advantage of the multi-wavelength optical phase unwrapping technique is that the beat wavelength can be tailored to match height variations of specific samples