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. 2010 Nov 3;132(43):15099-101.
doi: 10.1021/ja1044192.

Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores

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Free PMC article

Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores

Hsiao-lu D Lee et al. J Am Chem Soc. .
Free PMC article

Abstract

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.

Figures

Figure 1
Figure 1
The absorption of 1 in ethanol decreases during irradiation with 1.1 mW/cm2 at 385 nm shown at 15 s, 30 s, 90 s, and 150 s (left down arrow). Concurrently, the absorption of amine photoproduct 2 grows proportionally (center up arrow). The bright fluorescence from 2 when pumped at 594 nm is the dotted curve; the heavy dashed curve is dim fluorescence from the original sample of 1 (the small emission signal is most likely from the pre-activated amine contaminants in the azide sample). Preactivation can be minimized by keeping the samples in complete darkness. Compound 3 has the same photophysical properties as 1.
Figure 2
Figure 2
Evidence that the HaloTag-targeted fluorogen correctly labels specific proteins and enables SR imaging in mammalian cells. (A) Phase image of fixed WT HeLa cells. (B) The cells in A imaged in the DCDHFV-P channel. (C) The cells in A imaged in the Alexa488 channel. (D) Phase image of fixed HeLa expressing HaloEnz-α-tubulin labeled with 3. (E) The cells in D imaged in the DCDHF-V-P channel. (F) The cells in D imaged in Alexa488 channel. (G) Overlay of E, F, and additional blue DAPI channel to show nuclei. (H) Live CHO cells co-transfected to express both HaloEnz–α-tubulin and α-tubulin–eGFP labeled with 3 and imaged in DCDHF-V-P channel. (I) Cells from H imaged in the EGFP channel. (The higher background in H may be the result of nonspecific binding and imperfect washing of untargeted fluorophores.) (J) Fixed BS-C-1 cells expressing HaloEnz-α-tubulin labeled with 3 imaged using conventional diffraction-limited imaging. Indicated microtubule measures 450±40nm FWHM. (K) Same cell as J with SR imaging. Indicated microtubule measures 85±15nm FWHM. See SI for sample preparation procedures.
Figure 3
Figure 3
Diffraction-limited imaging of 4 inside live C. crescentus cells expressing fusion proteins to FtsZ and PopZ. These proteins localize as expected, indicating that the HaloTag–DCDHF labeling does not disrupt typical cellular behavior.
Figure 4
Figure 4
SR imaging of protein fusions inside live C. crescentus cells using 3. (A–C) PopZ forms a polymeric network at the poles of the cells. Compared to the DL images in Figure 3, these SR images reveal distinct shapes of the PopZ structure, including the cap-like network in C. (D–E) AmiC is recruited to the division plane early in the cell cycle. These SR images indicate that AmiC may form a structure that hugs the membrane. For details of imaging and image processing, see SI. The SR images are extracted from localizations over 75 seconds with a mean localization precision of 32 ± 12 nm.
Scheme 1
Scheme 1
Photochemical activation produces 2 from 1. A mixture of photoproducts is produced,, but the primary amine with R1=R2=H is the significant product (see Table 1 for reaction yield of primary amine). HaloTag versions of 1 and a separate fluorophore are also shown (3 and 4).

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