Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug 1;2(8):2364-71.
doi: 10.1364/BOE.2.002364. Epub 2011 Jul 22.

Two-color STED microscopy in living cells

Patrina A Pellett, Xiaoli Sun, Travis J Gould, James E Rothman, Ming-Qun Xu, Ivan R Corrêa Jr, Joerg Bewersdorf

Two-color STED microscopy in living cells

Patrina A Pellett et al. Biomed Opt Express. .

Abstract

Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells. Using improved SNAP(f) and CLIP(f) technologies to label epidermal growth factor (EGF) and EGF receptor (EGFR), we report resolutions of 78 nm and 82 nm for 22 sequential two-color scans in living cells.

Keywords: (170.3880) Medical and biological imaging; (180.2520) Fluorescence microscopy; (350.5730) Resolution.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic of experimental labeling procedure using SNAPf or CLIPf reactions to specifically label EGFR and EGF. (A) In living cells, SNAPf recognizes its substrate, BG, and undergoes a Sn2 type reaction that displaces BG, resulting in a permanent covalent bond between the EGFR and Chromeo494 (green star). (B) EGF-CLIPf is recombinantly expressed and purified, labeled with BC-ATTO647N (BC-647N) producing exogenous EGF-CLIPf-ATTO647N. (C) Live cells expressing EGFR-SNAPf are labeled with Chromeo494, followed by incubation with EGF-CLIPf-ATTO647N. This results in the double labeling of the EGFR-EGR complex for two-color STED imaging. EGFR is known to form a homodimer upon ligand binding, but is shown as a single receptor in this schematic for clarity. Cyto = cytoplasm, PM = plasma membrane and ECM = extracellular matrix.
Fig. 2
Fig. 2
Resolution measurements for ATTO647N and Chromeo494 in living HEK293 cells. HEK293 cells were treated with 100 ng/mL EGF-CLIPf-ATTO647N and imaged with (A) STED and (B) confocal microscopy. The area depicted by the white box (A) is shown in (C) STED and (D) confocal. The boxes in (C) and (D) were used to generate the line profiles for ATTO647N in (E) and (F). HEK293 cells stably expressing EGFR-SNAPf were treated with 10 μM BG-Chromeo494 for 10 min at 37°C and imaged by (G) STED and (H) confocal microscopy. The area depicted by the white box (G) for Chromeo494 is shown in (I) STED and (J) confocal. The boxes in (I) and (J) were used to generate the line profiles for Chromeo494 in (K) STED and (L) confocal. Solid red lines indicate the Lorentzian (STED) and Gaussian (confocal) least squares fits used to determine the full width at half-maximum (FWHM) values.
Fig. 3
Fig. 3
Live cell two-color STED time series of HEK293 cells labeled with EGF-CLIPf-ATTO647N (magenta) and EGFR-SNAPf-Chromeo494 (green). Data has been normalized to correct for bleaching. The shown images have been cropped from the original raw data. Scale bar = 1 μm.
See this image and copyright information in PMC

References

    1. Toomre D., Bewersdorf J., “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010). 10.1146/annurev-cellbio-100109-104048 - DOI - PubMed
    1. Hell S. W., Wichmann J., “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). 10.1364/OL.19.000780 - DOI - PubMed
    1. Westphal V., Rizzoli S. O., Lauterbach M. A., Kamin D., Jahn R., Hell S. W., “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008). 10.1126/science.1154228 - DOI - PubMed
    1. Hein B., Willig K. I., Wurm C. A., Westphal V., Jakobs S., Hell S. W., “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010). 10.1016/j.bpj.2009.09.053 - DOI - PMC - PubMed
    1. Hess S. T., Girirajan T. P. K., Mason M. D., “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006). 10.1529/biophysj.106.091116 - DOI - PMC - PubMed

LinkOut - more resources