Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones
| Autor: | Jérôme Boulanger, Atsushi Matsuda, Peter M. Carlton, Charles Kervrann, Lin Shao, Peter Kner, John W. Sedat, David A. Agard |
|---|---|
| Přispěvatelé: | Heintzmann, Rainer, The Keck Center for Advanced Microscopy, Department of Biochemistry and Biophysics [San Francisco], University of California, BioImaging Cell and Tissue Core Facility (PICT-IBiSA), Institut Curie [Paris], Space-timE RePresentation, Imaging and cellular dynamics of molecular COmplexes (SERPICO), Inria Rennes – Bretagne Atlantique, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), Howard Hughes Medical Institute [Chevy Chase] (HHMI), Howard Hughes Medical Institute (HHMI), University of California (UC) |
| Rok vydání: | 2010 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Fluorophore General Science & Technology Green Fluorescent Proteins Biophysics lcsh:Medicine Mitosis Bioengineering [SDV.BC]Life Sciences [q-bio]/Cellular Biology Fluorescence Chromosomes Histones 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Fluorescence microscope Animals Photoactivated localization microscopy lcsh:Science 030304 developmental biology 0303 health sciences Microscopy Multidisciplinary Chemistry Resolution (electron density) lcsh:R Biophysics/Structural Genomics Cell Biology Chromosomes Insect Autofluorescence Microscopy Fluorescence Chromosome Structures [INFO.INFO-TI]Computer Science [cs]/Image Processing [eess.IV] lcsh:Q Drosophila Generic health relevance Biological imaging Insect 030217 neurology & neurosurgery Research Article Biotechnology |
| Zdroj: | PloS one, vol 5, iss 9 PLoS ONE PLoS ONE, Public Library of Science, 2010, 5 (9), pp.e12768. ⟨10.1371/journal.pone.0012768⟩ PLoS ONE, Vol 5, Iss 9, p e12768 (2010) PLoS ONE, 2010, 5 (9), pp.e12768. ⟨10.1371/journal.pone.0012768⟩ |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0012768⟩ |
| Popis: | International audience; 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 redconverted 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|