Widely accessible method for superresolution fluorescence imaging of living systems
Widely accessible method for superresolution fluorescence imaging of living systems
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DOI：
10.1073/pnas.1204917109
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发表时间：
2012-07-03
影响因子：
11.1
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
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作者：
Dedecker, Peter;Mo, Gary C. H.;Zhang, Jin
超分辨率荧光显微镜克服了衍射分辨率的障碍，使生命的分子错综复杂的细节得以极大地增强。然而，目前的许多超分辨率技术仍然面临局限性，它们的实施通常与陡峭的学习曲线相关。基于图案化照明的超分辨技术[如受激辐射耗竭(STED)、可逆线性荧光跃迁(RESOLFT)和饱和结构照明显微镜(SSIM)]需要专门的设备，而基于单分子的方法[例如随机光学重建显微镜(STORM)、光激活定位显微镜(Palm)和荧光Palm(F-Palm)]涉及重复的单分子定位，这需要其自身的专业知识和时间上的要求。本文提出了一种超分辨荧光成像方法-光致变色随机光学涨落成像(PCSOFI)。在这种方法中，在适当的波长照射可逆光开关荧光蛋白产生健壮的单分子强度波动，可以通过统计分析每个像素中的波动作为时间的函数来提取超分辨率图像，正如之前在SOFI中所展示的那样。这种方法使用现成的设备、可遗传编码的标记和简单快速的数据采集，能够在活细胞中提供两到三倍增强的空间分辨率、显著的背景抑制、显著的对比度和良好的时间分辨率。此外，3D和多色成像都很容易实现。由于其易用性和高性能，我们预计PCSOFI将被证明是一种有吸引力的超分辨率成像方法。
Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.