condensed mitotic chromosome structure at nanometer resolution using palm and egfp- histones浓缩的有丝分裂染色体结构纳米分辨率使用palm和egfp -组蛋白.pdfVIP

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones 1¤a 1¤b 2 3 1¤d Atsushi Matsuda , Lin Shao , Jerome Boulanger , Charles Kervrann , Peter M. Carlton , Peter 1¤c 4 1 Kner , David Agard , John W. Sedat * 1 Department of Biochemistry and Biophysics, The Keck Center for Advanced Microscopy, University of California San Francisco, San Francisco, California, United States of America, 2 Institut Curie, Centre de Recherche, Paris, France, 3 INRIA Rennes Bretagne Atlantique, Campus Universitaire de Beaulieu, Rennes, France, 4 Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, The Keck Center for Advanced Microscopy, University of California San Francisco, San Francisco, California, United States of America Abstract 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 o