SummaryA key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response—in the absence of any amplification step—remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or “assisted” loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.
While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, we have used super-resolution microscopy to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. We find that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. We find that foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription.
We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views, in combination with a bespoke fitting algorithm, enable the 3D positions of single fluorophores to be determined from parallax. SMLFM achieves up to 20 nm localization precision throughout an extended 6 µ m depth of field. The capabilities of SMLFM are showcased by imaging membranes of fixed eukaryotic cells and DNA nanostructures below the optical diffraction limit.
We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm lateral and axial precision across a 6 µm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.
We propose a novel high-efficiency no-moving-parts Laguerre-Gauss (LG) spectrometer using two variable focus lenses and a variable sized pinhole that overcomes the limitations of the classical, projective, phase flattening technique for measuring the Laguerre-Gauss (LG) spectrum of light beams. Simulation results show that the coupling losses are virtually zero and the only losses are ring losses which are mode-dependent but beam waist-independent. Hence, the detection efficiency for all modes is simultaneously the maximum possible irrespective of the beam waist of the LG modes chosen for the decomposition. The losses can also be easily pre-calibrated to remove the efficiency bias amongst different modes. [4][5][6] as well as the OAM spectrum of light beams [7][8][9]. The measurement of LG spectrum of an unknown beam, though, remains tricky and direct methods such as projective phase-flattening [10] and then coupling into a single mode fiber (SMF) have their limitations [11]. In this paper, we address some of the limitations of the projective phase-flattening approach using electronically controlled variable focus lenses. Such variable focus lenses have been used in a number of applications [12].The projective phase-flattening approach works by projecting an unknown, incoming beam onto a conjugate Laguerre-Gaussian (LG) mode using a phase spatial light modulator (SLM). A Fourier lens is then used to take the Fourier transform of the resultant field at the phase SLM in the focal plane of the lens. This Fouriertransformed field is then coupled into a single-mode-fiber (SMF). If the input beam contains that particular mode, then the helical phase of the input beam is completely canceled and the Fourier-transformed field has a central bright spot similar to a Gaussian with a ringed intensity pattern around it. The central bright spot can then couple into an SMF as the SMF supports only the T EM 00 mode which is also similar to a Gaussian. The process is repeated for different modes to determine the complete LG spectrum.The limitations of this approach are highlighted in Ref. [11]. Firstly, the detection efficiency into the SMF varies from mode to mode and also with the selected value of beam waist w 0 of the LG mode (see Ref [11], Fig. 2). The maximum possible detection efficiency for all the different possible modes is not obtained for one particular value of w 0 , which implies that all modes cannot opti- * mumtaz.sheikh@lums.edu.pk mally couple into the SMF simultaneously. Moreover, for a particular value of w 0 , the detection efficiency decreases with mode order thus limiting the bandwidth of the measured OAM spectrum. Ideally, the choice of w 0 should be such that it gives high enough detection efficiencies for all modes. Secondly, the radial decomposition of the beam depends upon the value of the beam waist w 0 chosen for the modes. Optimal choice of the beam waist is the one that gives the minimum number of radial modes. However, that cannot be known a priori for an unknown incoming beam. Finally, since the...
Super-resolution techniques that localize single molecules in three dimensions through point spread function (PSF) engineering are very sensitive to aberrations and optical alignment. Here we show how double-helix point spread function is affected by such mis-alignment and aberration. Specifically, we demonstrate through simulation and experiment how misplacement of phase masks in infinity corrected systems is a common source of significant loss of accuracy. We also describe an optimal alignment and calibration procedure to correct for these errors. In combination, these optimizations allow for a maximal field of view with high accuracy and precision. Though discussed with reference to double-helix point spread function (DHPSF), the optimization techniques are equally applicable to other engineered PSFs.
Pseudomonas aeruginosa is highly successful in colonizing in all types of environments. P. aeruginosa colonizing in adverse environment due to the presence of its virulence factors include production of toxins, proteases hemolysins, and formation of biofilms. In man, the most common opportunist pathogen is P. aeruginosa. Metabolically P. aeruginosa is versatile. Most of the antibiotics targeted metabolically active cells and bacteria could contribute to decrease in biofilm susceptibility to the antimicrobial agents. Scientists suggested about Pseudomonas that it can be catabolized any hydrocarbon in specific time along with availability of oxygen and nitrite. If bacteria are not susceptible to one agent in three or more, it is called as multidrug-resistance strains. The antimicrobial treatments were not suitable when microorganism presented in vitro microorganism resistance to antimicrobials used for treatment of the patient which lack of treatment for 24 h after diagnosis of microbial infections. Bacteria have developed resistance against commonly used antibiotics. Treatment of Pseudomonas infections is coming harder day by day as its resistance against most of the antibiotics. Because of resistance of bacteria antibiotics, alternative methods are in consideration. These methods include use of lactic acid bacteria (LAB) and most recently nano-particles. That is why they are used as antibacterial agents.
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