Angewandte
Chemie
DOI: 10.1002/anie.201410240
Fluorescent Probes
Fluorescence Microscopy Visualization of Contacts Between Objects**
Tomislav Suhina, Bart Weber, Chantal E. Carpentier, Kinga Lorincz, Peter Schall, Daniel Bonn,*
and Albert M. Brouwer*
Abstract: The area of contact between two objects was detected
by using the strong enhancement of the fluorescence of
rigidochromic probe molecules attached to one of the surfaces.
Confinement of the molecules suppresses nonradiative decay
and turns on the fluorescence. The approach is demonstrated
by imaging of the contact area of a plastic sphere in contact
with a flat glass surface. Our results agree excellently with the
prediction of Hertzꢀs classical theory based on elastic defor-
mation.
decay is triggered by the rotation around a specific bond in
the molecule. When the rotation of the bond is hindered, the
non-radiative decay is suppressed, and the excited state
decays by emitting a photon. When rigidochromic molecules
are incorporated in a very viscous medium, such as a glassy
polymer matrix, a strong fluorescence is observed. This effect
has been used to measure local viscosities in polymer films
and study their free volume and glass transition,[2,5,7,8] and to
investigate the viscosity of membranes and intracellular
media.[3,9–11] We show that the confinement between two
surfaces also impedes the non-radiative relaxation of the
probe molecule 1 that starts fluorescing strongly when
confined. This effect then allows the detection of the physical
contacts between surfaces on a molecular scale.
T
he study of contact mechanics dates back to 1882 with the
publication of “On the contact of elastic solids” by Hertz.[1] For
the behavior of virtually all mechanical systems, the mechan-
ics of the contact between their constituents is crucial.
Friction, for instance, is a direct consequence of contact
mechanics and is responsible for about 30% of the world
energy consumption.[2] Surprisingly little is known about how
the physical contacts between objects arise, although this is
essential for understanding their mechanics.[3] The main
challenge is that since most (if not all) surfaces possess
a certain roughness, the actual contacts may occur on
microscopic length scales, even for large macroscopic
bodies. Bowden and Tabor were the first to emphasize the
importance of surface roughness for bodies in contact.[4]
Herein we describe the first direct visualization of
mechanical contacts at the microscale by means of fluores-
cence microscopy, using specifically developed probe mole-
cules that fluoresce when confined in a contact. To achieve
this goal we synthesized rigidochromic fluorescent molecules
that fluoresce only very weakly in (low-viscosity) solutions
owing to the presence of rapid non-radiative relaxation
pathways for the excited state.[5–7] This fast non-radiative
For our experiments, we synthesized a new member of the
DCDHF class of compounds that has in recent years been
developed by Moerner, Twieg, and co-workers for single-
molecule imaging.[8,9,11,14] (1, Scheme 1; for details, see the
Scheme 1. The compounds investigated in this study.
Supporting Information). This chromophore has advantages
over previously used viscosity sensitive probes such as
dicyanovinyljulolidines[6,7,15] and BODIPY dyes:[10,16,17] exci-
tation and emission in the visible part of the spectrum, good
photostability,[8] and particularly low fluorescence in low-
viscosity polar solvents. Compound 1 was chemically linked to
the surfaces of glass coverslips to investigate the imaging of
contact areas. Compound 2 was used for comparison.
[*] T. Suhina,[+] Dr. C. E. Carpentier,[+] Prof. Dr. A. M. Brouwer
van ’t Hoff Institute for Molecular Sciences
University of Amsterdam
P.O. Box 94157, 1090 GD Amsterdam (The Netherlands)
E-mail: a.m.brouwer@uva.nl
T. Suhina,[+] B. Weber,[+] Dr. C. E. Carpentier,[+] Dr. K. Lorincz,[+]
Prof.Dr. P. Schall, Prof. Dr. D. Bonn
As a first step to characterize their photophysical proper-
ties, we measured absorption and emission spectra of com-
pounds 1 and 2 in a series of solvents. The data listed in the
Supporting Information (Table S1) show that there is little
difference in the properties of 1 and 2, as expected. Both show
a weak solvatochromic effect in absorption and in emission.
The fluorescence quantum yields Ff are low, and tend to
decrease with increasing solvent polarity. Fluorescence decay
times tf follow the same trends as the quantum yields. In some
solvents they were shorter than the time resolution of our
instrument (< 10 ps). The quantum yields and decay times are
larger in solvents of higher viscosity. For example, in cyclo-
Institute of Physics, University of Amsterdam
P.O. Box 94485, 1090 GL Amsterdam (The Netherlands)
E-mail: d.bonn@uva.nl
[+] These authors contributed equally to this work.
[**] This work is part of the research program of the Foundation for
Fundamental Research on Matter (FOM), which is part of the
Netherlands Organisation for Scientific Research (NWO). Michiel
Hilbers helped with the confocal microscopy and fluorescence
decay time measurements. Table of contents graphic: Berta Garcia
Landa.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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