Optimizing imaging speed and excitation intensity for single-molecule localization microscopy

被引：70

作者：

Diekmann, Robin ^{[1
]}

Kahnwald, Maurice ^{[1
,2
]}

Schoenit, Andreas ^{[1
]}

Deschamps, Joran ^{[1
]}

Matti, Ulf ^{[1
]}

Ries, Jonas ^{[1
]}

机构：

[1] European Mol Biol Lab EMBL, Cell Biol & Biophys Unit, Heidelberg, Germany

[2] Friedrich Miescher Inst Biomed Res, Basel, Switzerland

来源：

NATURE METHODS | 2020年 / 17卷 / 09期

基金：

美国国家卫生研究院; 欧洲研究理事会;

关键词：

OPTICAL RECONSTRUCTION MICROSCOPY; RESOLUTION; FLUOROPHORES; NANOSCOPY; TRACKING;

D O I：

10.1038/s41592-020-0918-5

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

High laser powers are common practice in single-molecule localization microscopy to speed up data acquisition. Here we systematically quantified how excitation intensity influences localization precision and labeling density, the two main factors determining data quality. We found a strong trade-off between imaging speed and quality and present optimized imaging protocols for high-throughput, multicolor and three-dimensional single-molecule localization microscopy with greatly improved resolution and effective labeling efficiency.

引用

页码：909 / +

页数：22

共 30 条

[1] Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes [J].

Balzarotti, Francisco ;

Eilers, Yvan ;

Gwosch, Klaus C. ;

Gynna, Arvid H. ;

Westphal, Volker ;

Stefani, Fernando D. ;

Elf, Johan ;

Hell, Stefan W. .

SCIENCE, 2017, 355 (6325) :606-612

[2]

Barentine A. E. S., 2022, 3D MULTICOLOR NANOSC, DOI [10.1101/606954, DOI 10.1101/606954]

[3] Imaging intracellular fluorescent proteins at nanometer resolution [J].

Betzig, Eric ;

Patterson, George H. ;

Sougrat, Rachid ;

Lindwasser, O. Wolf ;

Olenych, Scott ;

Bonifacino, Juan S. ;

Davidson, Michael W. ;

Lippincott-Schwartz, Jennifer ;

Hess, Harald F. .

SCIENCE, 2006, 313 (5793) :1642-1645

[4] Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species [J].

Bossi, Mariano ;

Foelling, Jonas ;

Belov, Vladimir N. ;

Boyarskiy, Vadim P. ;

Medda, Rebecca ;

Egner, Alexander ;

Eggeling, Christian ;

Schoenle, Andreas ;

Hell, Stefan W. .

NANO LETTERS, 2008, 8 (08) :2463-2468

[5]

Dempsey GT, 2011, NAT METHODS, V8, P1027, DOI [10.1038/NMETH.1768, 10.1038/nmeth.1768]

[6] Photoswitching Mechanism of Cyanine Dyes [J].

Dempsey, Graham T. ;

Bates, Mark ;

Kowtoniuk, Walter E. ;

Liu, David R. ;

Tsien, Roger Y. ;

Zhuang, Xiaowei .

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2009, 131 (51) :18192-+

[7] Major signal increase in fluorescence microscopy through dark-state relaxation [J].

Donnert, Gerald ;

Eggeling, Christian ;

Hell, Stefan W. .

NATURE METHODS, 2007, 4 (01) :81-86

[8] 3D single-molecule super-resolution microscopy with a tilted light sheet [J].

Gustavsson, Anna-Karin ;

Petrov, Petar N. ;

Lee, Maurice Y. ;

Shechtman, Yoav ;

Moerner, W. E. .

NATURE COMMUNICATIONS, 2018, 9

[9] Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes [J].

Heilemann, Mike ;

van de Linde, Sebastian ;

Schuttpelz, Mark ;

Kasper, Robert ;

Seefeldt, Britta ;

Mukherjee, Anindita ;

Tinnefeld, Philip ;

Sauer, Markus .

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2008, 47 (33) :6172-6176

[10] Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution [J].

Huang, Bo ;

Jones, Sara A. ;

Brandenburg, Boerries ;

Zhuang, Xiaowei .

NATURE METHODS, 2008, 5 (12) :1047-1052

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