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. 2012 Dec;9(12):1181-4.
doi: 10.1038/nmeth.2214. Epub 2012 Oct 28.

Ultrabright photoactivatable fluorophores created by reductive caging

Joshua C Vaughan  1 , Shu Jia, Xiaowei Zhuang
Affiliations

Ultrabright photoactivatable fluorophores created by reductive caging

Joshua C Vaughan et al. Nat Methods. 2012 Dec.

Abstract

Sub-diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, for which the image resolution depends on the number of photons detected per localization. We report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 10(4)-10(6) photons per localization and allow imaging of fixed samples with resolutions of several nanometers. This strategy can be applied to many fluorophores across the visible spectrum.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Bright photoactivatable dyes created by reductive caging. (a) Left panel, initial fluorescence signal of a fixed cell immunolabeled for microtubules with Cy3B. Middle panel, fluorescence signal after reduction with NaBH4. Right panel, fluorescence signal after illumination with UV light, showing approximately 40% recovery of the initial fluorescence. The image contrast in the middle and right panels is multiplied by 2.5 compared to the left panel. Scale bar, 5 μm. (b) Fluorescence trace of a single Cy3B molecule recovering to a bright state after reduction and subsequent photoactivation (blue box). (c) Mean number of photons detected from individual Atto488, Cy3, Cy3B, Alexa 647, and Cy5.5 molecules after photoactivation. (d) Mean localization precision (left axis) and potentially obtainable resolution (right axis) determined from at least 100 molecules for each dye. Here, each molecule was tracked for many consecutive camera frames and a distribution of localizations from individual frames were constructed. The standard error of the mean (SEM) of the localization distribution for each molecule’s trajectory corresponds to the localization precision of each molecule when all photons detected from the molecule were used for a single localization. The potentially obtainable resolution, i.e. the minimum distance required to resolve two molecules, is determined as 2.35 times the localization precision assuming a Gaussian distribution.
Figure 2
Figure 2
STORM images of microtubules in cells stained by indirect immunofluorescence with (a) Atto 488, (b) Cy3B, and (c) Cy5.5. Samples were reduced in situ with NaBH4, washed, and then imaged with 488 nm, 561 nm, and 647 nm excitation light in a buffer designed to reduce blinking and photobleaching. Illumination with 405 nm light allowed control over the activation rate. Scale bar, 1 μm.
Figure 3
Figure 3
STORM image of microtubules polymerized and labeled in vitro with Cy3B. (a) A STORM image of microtubules (green) with several magnified zoom-in images shown in insets. A portion of the corresponding conventional fluorescence image (magenta) is overlaid on the STORM image. (b) Transverse cross-sectional profiles of the boxed microtubule segments (i)–(iii). Segments (i) and (ii) show hollow microtubule profiles with 16–18 nm inner diameter. The red curves are nonlinear least-squares fits of the distribution to two Gaussian functions. Segment (iii) shows a segment of microtubules that are less well-resolved, which gives a top-hat profile with an overall width of 26.6 nm. Scale bars, 1 μm in the main image and 100 nm in all image insets.
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