Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy
J Fluoresc
Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy
Christian M. Winterflood 0 1
Stefan Seeger 0 1
0 Department of Chemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich , Switzerland
1 Randall Division of Cell and Molecular Biophysics, King's College London , London SE1 1UL , UK
2 Christian M. Winterflood
We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.
Fluorescence correlation spectroscopy; Supercritical angle fluorescence; Undercritical angle fluorescence; Surface-selective; Near-field; Far-field
Introduction
For the study of processes at surfaces and interfaces the
standard confocal FCS has the immanent problem that the
ellipsoidal observation volume suffers from having a low axial
confinement. As a result, surface processes remain concealed
by the background produced by the bulk fluorescence.
Optical near fields have been succefully used to confine
observation volumes to interfaces. FCS has, for instance,
been performed using evanescent waves produced at
optical nanostructures called zero-mode waveguides [
1–3
] or
more commonly using TIRF [
4–7
]. TIR-FCS uses
objective-type TIRF illumination to restrict the excitation
to a thin section less than 200 nm above the interface in
combination with standard confocal detection to ensurse
the lateral confinement of the detection volume. TIR-FCS
has proven very useful for the study of processes close to a
surface/solution interface. In theory, it can give access to a
number of properties, including local fluorophore
concentrations and local fluorophore translational mobility [8], or
kinetic rate constants for reversible association of
fluorophores with the interface [
9
]. The determination of
these quantities by TIR-FCS, however, relies on the a priori
knowledge of the fluorescent solution concentration. In
many biological cases, though, such as the study of the
interaction of proteins with membranes or membrane
proteins, rely on the use of fluorescent fusion proteins whose
cellular expression levels are not precisely known [
10
].
While the advantages of SAF-CS have already been
described [
11
], in this report we provide an extension to the
technique which allows to perform FCS in close proximity
to the sample/solution interface as well as deeper in
solution simultaneously.
Results
We use make use of a previously reported custom setup and
microscope objective [
12
] (Fig. 1) for tighly focused,
undercritical angle excitation and parallel, well-separated
collection of SAF and UAF. SAF collection yields a highly
surface-confined detection volume, while UAF collection
yields a conventional confocal volume which extends deeper
into the sample. The simultaneous measurement of SAF and
UAF has been used for determining axial emitter positions
with nanometer accuracy [
13
] as well as to reduce artifacts
in membrane FCS related to a non-planar geometry of the
membrane [
14
].
Quantitive results in FCS rely on the size and shape of
the detection volume. The most common way of calibrating
the detection volume is to perform FCS on a fluorescent
species with known diffusion coefficient and concentration.
While the temporal decay of the autocorrelation function
(ACF) depends on the shape of the observation volume,
the amplitude of the ACF gives direct access to the size of
the detection volume through the relationship Veff = 1/
(G0×C). Here, Veff is the socalled effective volume, G0 the
amplitude of the ACF, and C the concentration of the
sample. In turn, it is possible to determine concentrations of
fluorescent species with a calibrated effective volume. We
carried out diffusion measurements on the red fluorescent
dye Atto655 (in its carboxylic acid form, −COOH) which
has negligible triplet state contributions and a precisely
determined diffusion coefficient [
15
]. A difficulty when
trying to probe the detection volume at the coverslip/solute
interface by free diffusion arises from non-specific
interaction of the fluorophore with the coverslip glass. This flaws
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