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

NEW METHODS FOR FCS: FLUORESCENCE LIFETIME CORRELATION
SPECTROSCOPY AND TWO-FOCUS FCS
S. Orthaus, B. Krämer, V. Buschmann, P. Kapusta, U. Ortmann, M. König, F. Koberling,
A. Bülter, and R. Erdmann
PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
orthaus@picoquant.com
Ultrasensitive fluorescence detection and spectroscopy is important in many fields of
fundamental research as well as chemo- and bioanalytical applications. In recent years,
technical improvements in photodetector sensitivity, microscope objective optics, and laser
light sources have enhanced the capabilities for the detection of single molecules. This
technique allows to visualize variations from molecule to molecule which would be hidden
performing ensemble measurements. Today, time-resolved measurements permit to follow
fluorescence dynamics of single molecules starting in the sub-nanosecond range up to
fluctuations in the second range and beyond. By exploiting the full information content of
such a multi-dimensional measurement, classical intensity based analysis schemes like FCS in
confocal microscopy can be significantly improved by sorting and weighting the detected
photons. We will present actual instrumentation and discuss recent applications:
� Fluorescence Lifetime Cross Correlation Spectroscopy (FLCCS):
Fluorescence cross correlation spectroscopy (FCCS) is a superior tool to detect
binding between two molecules, each marked with a different fluorophore, in liquid
environment. Concentrations ranging from pM to µM can be investigated. Only bound
molecules moving together through the femtoliter detection volume contribute to the
cross correlation amplitude and are quantified.
However, artefacts like spectral bleed through are compromising the detection
sensitivity of bound molecules. To overcome this limitation, the combination of FCCS
with fluorescence lifetime measurements allows to suppress bleed through as well as
common parasitic contributions like Raman scattering and detector afterpulsing [1].
� Two-Focus FCS (2fFCS):
Small structural changes of molecules (e.g. proteins) can be investigated in their
natural environment by determining the diffusion coefficient. The necessary accuracy
for measuring the molecular hydrodynamic radius down to some ångström is met with
Two-Focus FCS.
Two orthogonally polarized laser beams pulsed in an alternating fashion (Pulsed-
Interleaved Excitation, PIE) are used to generate a robust dual foci geometry with a
well known focal distance. This intrinsic length scale refines diffusion studies in
solution and allows to overcome various uncertainties formerly relying on the size and
shape of the confocal volume in single focus FCS. It also dramatically improves the
accuracy of determining absolute diffusion coefficients [2].
References:
[1] P. Kapusta, M. Wahl, A. Benda, M. Hof, J. Enderlein, “Fluorescence Lifetime Correlation
Spectroscopy”, J. Fluorescence, 17, 043-048 (2007)
[2] T. Dertinger, A. Loman, B. Ewers, C. B. Müller, B. Krämer, J. Enderlein, “The Optics and
Performance of Dual-Focus Fluorescence Correlation Spectroscopy”, Optics Express, 16,
14353-14368 (2008)