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Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent mole...

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Autores principales: Matsuda, Atsushi, Shao, Lin, Boulanger, Jerome, Kervrann, Charles, Carlton, Peter M., Kner, Peter, Agard, David, Sedat, John W.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939896/
https://www.ncbi.nlm.nih.gov/pubmed/20856676
http://dx.doi.org/10.1371/journal.pone.0012768
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author Matsuda, Atsushi
Shao, Lin
Boulanger, Jerome
Kervrann, Charles
Carlton, Peter M.
Kner, Peter
Agard, David
Sedat, John W.
author_facet Matsuda, Atsushi
Shao, Lin
Boulanger, Jerome
Kervrann, Charles
Carlton, Peter M.
Kner, Peter
Agard, David
Sedat, John W.
author_sort Matsuda, Atsushi
collection PubMed
description Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.
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spelling pubmed-29398962010-09-20 Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones Matsuda, Atsushi Shao, Lin Boulanger, Jerome Kervrann, Charles Carlton, Peter M. Kner, Peter Agard, David Sedat, John W. PLoS One Research Article Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples. Public Library of Science 2010-09-15 /pmc/articles/PMC2939896/ /pubmed/20856676 http://dx.doi.org/10.1371/journal.pone.0012768 Text en Matsuda et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Matsuda, Atsushi
Shao, Lin
Boulanger, Jerome
Kervrann, Charles
Carlton, Peter M.
Kner, Peter
Agard, David
Sedat, John W.
Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
title Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
title_full Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
title_fullStr Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
title_full_unstemmed Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
title_short Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
title_sort condensed mitotic chromosome structure at nanometer resolution using palm and egfp- histones
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939896/
https://www.ncbi.nlm.nih.gov/pubmed/20856676
http://dx.doi.org/10.1371/journal.pone.0012768
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