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. 2009 Sep 8;48(35):8279-81.
doi: 10.1021/bi900870u.

A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling

Rita L Strack  1 , Birka Hein, Dibyendu Bhattacharyya, Stefan W Hell, Robert J Keenan, Benjamin S Glick
Affiliations
Free PMC article

A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling

Rita L Strack et al. Biochemistry. .
Free PMC article

Erratum in

  • Biochemistry. 2009 Oct 13;48(40):9704

Abstract

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy.

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Figures

Figure 1
Figure 1
Fluorescence properties of E2-Crimson. (A) Excitation spectrum. (B) Emission spectrum. (C) Photobleaching kinetics of E2-Crimson and other representative far-red FPs during widefield fluorescence microscopy with a Texas Red filter set. (D) Maturation kinetics of E2-Crimson and other representative far-red FPs. Data for panels C and D are the means of three independent measurements.
Figure 2
Figure 2
E2-Crimson is noncytotoxic to HeLa cells under conditions of standard high-level expression. HeLa cells were transiently transfected for constitutive high-level expression of the indicated FP. Three wells per FP were analyzed daily by flow cytometry. The average brightness of viable fluorescent cells was measured, and the strongest signal for a given FP was normalized to 100 units. Error bars represent sem.
Figure 3
Figure 3
E2-Crimson is useful for multicolor and STED microscopy. (A) A three-color yeast strain was generated by labeling the ER with E2-Crimson, late Golgi cisternae with GFP, and the cytoplasm with E2-Orange. The scale bar is 3 μm. (B) The mammalian ER was imaged by conventional confocal microscopy (left) or by STED microscopy (right) with 635 nm excitation and a STED wavelength of 760 nm. Fluorescence from the region designated by white dashed lines is quantified in Figure S7. The scale bar is 1 μm.
See this image and copyright information in PMC

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