12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

SUPER-RESOLUTION FLUORESCENCE IMAGING
WITH STANDARD FLUOROPHORES
Sebastian van de Linde*, Anindita Mukherjee*, Mark Schüttpelz**,
,
Steve Wolter**
, Mike Heilemann**
, Markus Sauer*
**
* Biotechnology & Biophysics, Julius-Maximilians-University Würzburg, Am Hubland,
97074 Wuerzburg, Germany
** Applied Laser Physics & Laser Spectroscopy, Bielefeld University, Universitaetsstr. 25,
33615 Bielefeld, Germany
sauer@physik.uni-bielefeld.de
Fluorescence microscopy has become an essential tool in biological and biomedical sciences
for imaging of living cells and tissues. In contrast to other imaging techniques, such as
electron microscopy, fluorescence microscopy offers an advantage because it enables imaging
even of dynamic processes in living cells. The weakness, however, has been the fact that the
spatial resolution was limited to ~ 200 nm in the imaging plane for more than 100 years [1].
Only recently methods emerged that enable subdiffraction resolution imaging based on
photoswitchable or photoactivatable fluorescent proteins and organic fluorophores [2].
Currently, most super-resolution approaches control fluorescence emission of fluorophores by
modulating the transition of fluorophores between a fluorescent (on) and non-fluorescent (off)
state. However, super-resolution fluorescence imaging with commercially available small
organic fluorophores remains challenging.
Here we introduce a novel and facile method that enables super-resolution imaging with
standard Alexa Fluor and ATTO dyes. We demonstrate that these frequently used
fluorophores which span the visible wavelength range from 480 to 700 nm can be switched
reversibly between an on- and off-state under similar experimental conditions. The recipe we
developed comprises solely the addition of thiol-containing reducing agents such as ßmercaptoethylamine
(MEA), dithiothreitol (DTT), or glutathione (GSH) which efficiently
populate a relatively stable non-fluorescent state. Since oxygen does not have to be removed
and reducing agents such as glutathione are present in the cytoplasm of cells in the µM to mM
range the method we describe is very relevant for super-resolution imaging with molecular–
scale resolution (~ 20 nm) even in living cells [3].
[1] E. Abbe, Arch Mikrosk Anat 9, 413 (1873).
[2] M. Heilemann, P. Dedecker, J. Hofkens, M. Sauer, Laser and Photonics Review 3, 180
(2009).
[3] M. Heilemann, S. van de Linde, A. Mukherjee, M. Sauer, Angew Chem Int Ed. (2009).
12 th <strong>Zeiss</strong> <strong>sponsored</strong> FCS <strong>workshop</strong> – Cargèse, Corsica, France – <strong>12th</strong> -16th October 2009