Ingo Gregor

Art and Artefacts of Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is an important technique for studying low concentrations of analyte molecules in solution. The core molecular characteristic that can be addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for i.e. studying molecular binding and reactions, or conformational changes of macromolecules. The present paper discusses several possible optical and photophysical effects that can influence the outcome of a FCS measurement and thus can bias the value of the derived diffusion coefficient.

Photoluminescence of carbon nanodots: dipole emission centers and electron-phonon coupling.

Inorganic carbon nanomaterials, also called carbon nanodots, exhibit a strong photoluminescence with unusual properties and, thus, have been the focus of intense research. Nonetheless, the origin of their photoluminescence is still unclear and the subject of scientific debates. Here, we present a single particle comprehensive study of carbon nanodot photoluminescence, which combines emission and lifetime spectroscopy, defocused emission dipole imaging, azimuthally polarized excitation dipole scanning, nanocavity-based quantum yield measurements, high resolution transmission electron microscopy, and atomic force microscopy. We find that photoluminescent carbon nanodots behave as electric dipoles, both in absorption and emission, and that their emission originates from the recombination of photogenerated charges on defect centers involving a strong coupling between the electronic transition and collective vibrations of the lattice structure.

Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting

Fluorescence correlation spectroscopy (FCS) is a powerful spectroscopic technique for studying samples at dilute fluorophore concentrations down to single molecules. The standard way of data acquisition, at such low concentrations, is an asynchronous photon counting mode that generates data only when a photon is detected. A significant problem is how to efficiently convert such asynchronously recorded photon count data into a FCS curve. This problem becomes even more challenging for more complex correlation analysis such as the recently introduced combination of FCS and time-correlated single-photon counting (TCSPC). Here, we present, analyze, and apply an algorithm that is highly efficient and can easily be adapted to arbitrarily complex correlation analysis.

Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. Usually, FCS is measured with sensitive light detectors working in the photon-counting Geiger mode. A common property of such detectors is afterpulsing: the generation of spurious photon detection events after a genuine photon detection. Such afterpulsing causes a significant deviation of the measured autocorrelation function from its true value on a short time scale and can seriously influence derived parameters for fast processes such as triplet-state photophysics. Here, we discuss the impact of afterpulsing on FCS in detail. A new method is developed to eliminate afterpulsing effects by using time-correlated single-photon counting for separating the true fluorescence signal from afterpulsing events.

The rate of change in Ca2+ concentration controls sperm chemotaxis

Sperm navigate in a chemoattractant gradient by translating changes in intracellular calcium concentration over time into changes in curvature of the swimming path.

Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosomes catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.

Multi-target spectrally resolved fluorescence lifetime imaging microscopy

We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy

We present a thorough introduction into the recently developed fluorescence lifetime correlation spectroscopy (FLCS). The theoretical basis of FLCS is explained, and the method is applied to the study of a dynamic transition between two fluorescence lifetime states in a dye-protein complex.

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels

Time-correlated single photon counting is a powerful method for sensitive time-resolved fluorescence measurements down to the single molecule level. The method is based on the precisely timed registration of single photons of a fluorescence signal. Historically, its primary goal was the determination of fluorescence lifetimes upon optical excitation by a short light pulse. This goal is still important today and therefore has a strong influence on instrument design. However, modifications and extensions of the early designs allow for the recovery of much more information from the detected photons and enable entirely new applications. Here, we present a new instrument that captures single photon events on multiple synchronized channels with picosecond resolution and over virtually unlimited time spans. This is achieved by means of crystal-locked time digitizers with high resolution and very short dead time. Subsequent event processing in programmable logic permits classical histogramming as well as time tagging of individual photons and their streaming to the host computer. Through the latter, any algorithms and methods for the analysis of fluorescence dynamics can be implemented either in real time or offline. Instrument test results from single molecule applications will be presented.

Quantifying the Diffusion of Membrane Proteins and Peptides in Black Lipid Membranes with 2-Focus Fluorescence Correlation Spectroscopy

Protein diffusion in lipid membranes is a key aspect of many cellular signaling processes. To quantitatively describe protein diffusion in membranes, several competing theoretical models have been proposed. Among these, the Saffman-Delbrück model is the most famous. This model predicts a logarithmic dependence of a proteins diffusion coefficient on its inverse hydrodynamic radius (D ∝ ln 1/R) for small radius values. For large radius values, it converges toward a D ∝ 1/R scaling. Recently, however, experimental data indicate a Stokes-Einstein-like behavior (D ∝ 1/R) of membrane protein diffusion at small protein radii. In this study, we investigate protein diffusion in black lipid membranes using dual-focus fluorescence correlation spectroscopy. This technique yields highly accurate diffusion coefficients for lipid and protein diffusion in membranes. We find that despite its simplicity, the Saffman-Delbrück model is able to describe protein diffusion extremely well and a Stokes-Einstein-like behavior can be ruled out.