Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones.
Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones.
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DOI：
10.1371/journal.pone.0012768
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发表时间：
2010-09-15
期刊：
影响因子：
3.7
通讯作者：
Sedat JW
中科院分区：
文献类型：
--
作者：
Matsuda A;Shao L;Boulanger J;Kervrann C;Carlton PM;Kner P;Agard D;Sedat JW
光活化定位显微镜（PALM）和相关的荧光生物成像方法能够提供非常高的空间分辨率（高达20 nm）。两个主要的需求限制了其在生物样品上的广泛使用：对可光活化/可光转换的荧光分子的需求，其有时难以掺入，以及来自三维成像中相邻焦平面中的自体荧光或荧光团的高背景信号，其显著降低了PALM分辨率。我们在这里提出了一个高分辨率的PALM方法，利用传统的EGFP作为光转换荧光团，改进的算法来处理高水平的生物背景噪声，并将其应用于成像高阶染色质结构。我们发现，在还原核黄素存在下，当暴露于蓝光时，EGFP的发射波长有效地从绿色转换为红色。使用核黄素的红色转换的EGFP的光子产率与允许<20 nm分辨率的其他明亮的光转换荧光蛋白相当。我们进一步发现，使用原始PALM图像的去噪和去卷积的组合的图像预处理实质上提高了从噪声图像重建的空间分辨率。在用H2 AvD-EGFP（组蛋白H2 A变体）标记的果蝇有丝分裂染色体上进行PALM，显示出约70 nm的丝状组分。这是第一次在分辨率接近常规电子显微镜图像（10-30 nm）的情况下观察到一种组蛋白变体的细染色质细丝。正如在一个具有挑战性的标本上进行的建模和实验所证明的那样，这里描述的技术有助于普通生物样品的超分辨率荧光成像。
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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影响因子：
48
作者：
Biteen, Julie S.;Thompson, Michael A.;Tselentis, Nicole K.;Bowman, Grant R.;Shapiro, Lucy;Moerner, W. E.
通讯作者：
Moerner, W. E.
影响因子：
3.4
作者：
Aitken, Colin Echeverria;Marshall, R. Andrew;Puglisi, Joseph D.
通讯作者：
Puglisi, Joseph D.
影响因子：
3.4
作者：
HIRAOKA, Y;SEDAT, JW;AGARD, DA
通讯作者：
AGARD, DA
DOI：
10.1073/pnas.0810057105
发表时间：
2008-12-16
影响因子：
11.1
作者：
Eltsov, Mikhail;MacLellan, Kirsty M.;Dubochet, Jacques
通讯作者：
Dubochet, Jacques
DOI：
10.1073/pnas.0907866106
发表时间：
2009-12-29
影响因子：
11.1
作者：
Dertinger, T.;Colyer, R.;Enderlein, J.
通讯作者：
Enderlein, J.