Fluorescence Correlation Spectroscopy (FCS) has been invented more than 30 years ago and experienced a renaissance
after stable and affordable laser sources and low-noise single-photon detectors have become available. Its ability to
measure diffusion coefficients at nanomolar concentrations of analyte made it a widely used tool in biophysics.
However, in recent years it has been shown by many authors that aberrational (e.g. astigmatism) and photophysical
effects (e.g. optical saturation) may influence the result of an FCS experiment dramatically, so that a precise and reliable
estimation of the diffusion coefficient is no longer possible.
Here, we report on the development, implementation, and application of a new and robust modification of FCS that we
termed two-focus FCS (2fFCS) and which fulfils two requirements: (i) It introduces an external ruler into the
measurement by generating two overlapping laser foci of precisely known and fixed distance. (ii) These two foci and
corresponding detection regions are generated in such a way that the corresponding molecule detection functions
(MDFs) are sufficiently well described by a simple two-parameter model yielding accurate diffusion coefficients when
applied to 2fFCS data analysis.
Both these properties enable us to measure absolute values of the diffusion coefficient with an accuracy of a few percent.
Moreover, it turns out that the new technique is robust against refractive index mismatch, coverslide thickness
deviations, and optical saturation effects, which so often trouble conventional FCS measurements. Additionally, we will
show data that indicates that with 2fFCS it is even possible to monitor conformational changes of a calcium bindig
protein affecting the hydrodynamic radius by as little as two Angstrom.
We report on our application of a new fluorescence-correlation spectroscopy technique, 2-focus FCS, for measuring the
hydrodynamic radius of molecules with sub- Ångstrøm precision. The method is applied of monitoring conformational
changes of proteins upon ion binding. In particular, we present measurements on Ca2+-binding of recoverin. Recoverin
belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. The protein undergoes conformational changes upon Ca2+-changes that are reflected as changes in their hydrodynamic radius. By using 2fFCS
we were able to resolve hydrodynamic radius changes of ca. one Ångstrøm and used the Ca2+ dependence of this radius
for recording binding curves in solution. We compare our results with those obtained by other techniques.
A new concept of fluorescence microscopy is presented allowing the breaking of the diffraction limit of optical microscopy by a factor of ca. five. It relies on measuring the temporal evolution of fluorescence after sudden switch-on of the light excitation. The observed temporal dynamics of the fluorescence signal can be converted into information about the spatial distribution of fluorophores within the exciting laser focus. The proposed scheme is technically simple and versatile, and allows resolution enhancement in all three dimensions.
We present a new method for precisely measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations. The method is based on a modified Fluorescence Correlation Spectroscopy (FCS)-setup which is robust against many artifacts that are inherent to standard FCS 1, 2. The core idea of the new method is the introduction of an external ruler by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by ab initio calculations of resulting correlation curves and subsequent affine transformation of these curves to match the measured auto- and cross-correlation functions. The affine transformation coefficient along the time axis then directly yields the correct diffusion coefficient. This method is not relying on the rather inexact assumption of a 3D Gaussian shaped detection volume. We measured the diffusion coefficient of the red fluorescent dye Atto-655 (Atto-Tec GmbH) in water and compared the obtained value with results from Gradient Pulsed Field NMR (GPF-NMR).
A new multi-focus multi-confocal set-up for performing fluorescence spectroscopy of single molecules in solution is presented. The ultimate goal of the set-up is to track individual molecules during diffusion in solution, when all standard methods of trapping such as optical tweezers or dielectrophoretic traps fail. We present here a detailed description of the experimental setup and show first experimental results.
Fluorescence correlation spectroscopy (FCS) is an important technique for studying analyte molecules on a single molecule level in solution. The core molecular characteristic that is addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for studying molecular binding interactions or conformational changes of macromolecules. We present a thorough theoretical analysis of the FCS technique, paying special attention to the various frequently occurring technical artifacts. Particularly, we consider the influence of refractive index mismatch, cover-slide thickness, fluorescence anisotropy, optical adjustment, and optical saturation on the measured autocorrelation curve (ACF). The impact of these factors on the apparently determined diffusion coefficient is quantitatively evaluated. Extensive experimental results are presented demonstrating the theoretically predicted effects and dependencies.
We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 nm, 405 nm, 440 nm and 470 nm for fluorescence excitation which are coupled in through a sole single mode fiber. The detected signal is guided to a photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC5,7 system, via the unique Time-Tagged Time-Resolved (TTTR)6 data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently fit the full fluorescence lifetime image. The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows not only to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for the purpose of Fluorescence Lifetime Imaging (FLIM) or advanced Fluorescence Resonance Energy Transfer (FRET) analysis. We present here some selected results acquired with standard laser scanning microscopes upgraded for the time-correlated single photon counting technique.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.