Read e-book online Chromosome Architecture: Methods and Protocols PDF

By Mark C. Leake

This quantity information a precious number of protocols and stories, reminiscent of rising experimental and theoretical methods. those techniques have ended in a considerable development within the figuring out of chromosome architecture. Chromosome structure: equipment and Protocols courses readers via state-of-the-art interdisciplinary tools which enable for an figuring out of structure of chromosomes with quite superior answer, either when it comes to house and time. Written within the hugely profitable Methods in Molecular Biology series layout, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and tips about troubleshooting and keeping off identified pitfalls.

Authoritative and cutting-edge, Chromosome structure: equipment and Protocols aims to make sure profitable leads to the additional learn of this important field.

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Rather than standard epifluorescence illumination the laser can be inclined through the sample, by introducing an offset, d, in the position of the imaging laser at the BFP of the objective. Additionally, adding a slit in the conjugate plane to the specimen plane (HILO) can improve S/N further. (c) Schematic showing the two focal planes which must be imaged, the specimen plane containing the NPCs, and the fiducial plane where fluorescent beads can be utilized to correct lateral drift.  Individual NPCs can be identified as discrete puncta.

Two EMCCD cameras are attached to the camera port via a relay, enabling simultaneous two-color imaging. Cameras and lasers are synchronized using an external signal generator. An infrared laser autofocus system is coupled via the camera port to maintain a focus lock during image acquisition Single-Molecule Imaging to Characterize the Transport Mechanism of the Nuclear… 23 through an Acousto-Optical Tunable Filter (AOTF, AA Optoelectronics, France) to quickly modulate laser power. A. air) objective lens.

Autofluorescence can be further reduced using low fluorescence agarose, for example the Nusieve GTG Agarose from Lonza Biosciences. Ideal laser intensity settings will vary between microscope setups. It is recommended to use low laser intensities during imaging in order to reduce photobleaching or phototoxicity effects. The same applies for laser intensity used for pulse-­ bleaching. It is recommended to use an intensity that is high enough to completely bleach fluorescence in the region of interest, but not too high that other regions of the cell are bleached as well.

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