Fun: Single-molecule-fluorescence set-up-building for beginners…

The picture shows important characteristics of a state-of-the-art microscope: multicolour-excitation path with alternating laser excitation (blue, green, and red lasers),  a microscope stage with objective focusing the laser light into a small sample volume, the fluorescence from the sample volume is detected through the same objective, and the light is focussed onto a camera. For more information, click [here].

Publication: Characterization of Dark Quencher Chromophores as Nonfluorescent Acceptors for Single-Molecule FRET

L. Le Reste, J. Hohlbein, K. Gryte, A.N. Kapanidis, Biophysical Journal, 102, 11, 2658-2668, 2012, [link]

Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.

News: Hello Wageningen (NL)!

Good news: I accepted an offer for a position in the Laboratory of Biophysics in Wageningen (NL). Starting in Autumn 2012, my group is going to employ single-molecule methods, such as single‐molecule FRET and super‐resolution microscopy, to study biological processes on the molecular level.

We will study DNA-Protein interactions with a focus on DNA replication and repair. Besides continuing work on the bacterial DNA polymerase I (Klenow Fragment),  we will start investigating the human base excision repair pathway (BER), a cellular mechanism responsible for the repair of damaged sites in DNA.

If you are interested in joining the group as a Bachelor-, Master- or PhD-student, don’t hesitate to contact me for more information!

Publication: Identifying molecular dynamics in single-molecule FRET experiments with Burst Variance Analysis

J.P. Torella, S.J. Holden, Y. Santoso, J. Hohlbein, A.N. Kapanidis, Biophysical Journal, Vol. 100, Issue 6, 1568-1577, 2011, [link]

Histograms of single-molecule Förster resonance energy transfer (FRET) efficiency are often used to study the structures of biomolecules and relate these structures to function. Methods like probability distribution analysis analyze FRET histograms to detect heterogeneities in molecular structure, but they cannot determine whether this heterogeneity arises from dynamic processes or from the coexistence of several static structures. To this end, we introduce burst variance analysis (BVA), a method that detects dynamics by comparing the standard deviation of FRET from individual molecules over time to that expected from theory. Both simulations and experiments on DNA hairpins show that BVA can distinguish between static and dynamic sources of heterogeneity in single-molecule FRET histograms and can test models of dynamics against the observed standard deviation information. Using BVA, we analyzed the fingers-closing transition in the Klenow fragment of Escherichia coli DNA polymerase I and identified substantial dynamics in polymerase complexes formed prior to nucleotide incorporation; these dynamics may be important for the fidelity of DNA synthesis. We expect BVA to be broadly applicable to single-molecule FRET studies of molecular structure and to complement approaches such as probability distribution analysis and fluorescence correlation spectroscopy in studying molecular dynamics.

Publication: Defining the limits of single-molecule FRET resolution in TIRF microscopy

S.J. Holden, S. Uphoff, J. Hohlbein, D. Yadin, L. Le Reste, O.J. Britton, A.N. Kapanidis, Biophysical Journal, Vol. 99, Issue 9, 2011, 3102-3111 [link]

Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2–10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the limits of resolution of the technique, it is essential to investigate and rigorously quantify the major sources of noise and error; we used theoretical predictions, simulations, advanced image analysis, and detailed characterization of DNA standards to quantify the limits of TIRF-FRET resolution. We present a theoretical description of the major sources of noise, which was in excellent agreement with results for short-timescale smFRET measurements (<200 ms) on individual molecules (as opposed to measurements on an ensemble of single molecules). For longer timescales (>200 ms) on individual molecules, and for FRET distributions obtained from an ensemble of single molecules, we observed significant broadening beyond theoretical predictions; we investigated the causes of this broadening. For measurements on individual molecules, analysis of the experimental noise allows us to predict a maximum resolution of a FRET change of 0.08 with 20-ms temporal resolution, sufficient to directly resolve distance differences equivalent to one DNA basepair separation (0.34 nm). For measurements on ensembles of single molecules, we demonstrate resolution of distance differences of one basepair with 1000-ms temporal resolution, and differences of two basepairs with 80-ms temporal resolution. Our work paves the way for ultra-high-resolution TIRF-FRET studies on many biomolecules, including DNA processing machinery (DNA and RNA polymerases, helicases, etc.), the mechanisms of which are often characterized by distance changes on the scale of one DNA basepair.

Publication: Surfing on a new wave of single-molecule methods

J. Hohlbein, K. Gryte, M. Heilemann, A.N. Kapanidis, Phys. Biol., 7, 031001, 2010, [link]

Single-molecule fluorescence microscopy is currently one of the most popular methods in the single-molecule toolbox. In this review, we discuss recent advances in fluorescence instrumentation and assays: these methods are characterized by a substantial increase in complexity of the instrumentation or biological samples involved. Specifically, we describe new multi-laser and multi-colour fluorescence spectroscopy and imaging techniques, super-resolution microscopy imaging and the development of instruments that combine fluorescence detection with other single-molecule methods such as force spectroscopy. We also highlight two pivotal developments in basic and applied biosciences: the new information available from detection of single molecules in single biological cells and exciting developments in fluorescence-based single-molecule DNA sequencing

Publication: Conformational transitions in DNA polymerase I revealed by single-molecule FRET

Y. Santoso, C.M. Joyce, O. Potapova, L. Le Reste, J. Hohlbein, J.P. Torella, N.D.F. Grindley, and A.N. Kapanidis, PNAS, 107, 715, 2010, [link]

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond-timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.

Publication: Single-Molecule FRET: Methods and Biological Applications in Handbook of Single-molecule Biophysics

L.C. Hwang, J. Hohlbein, S.J. Holden, and A.N. Kapanidis. Springer-Verlag New York Inc., edit. P. Hinterdorfer and A. Van Oijen, 2009

Describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. This title includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, and enzymology and biochemistry.

Publication: Three-dimensional orientation determination of the emission dipoles of single molecules: The shot noise limit

J. Hohlbein, and C.G. Hübner, Journal of Chemical Physics, 129, 094703, 2008, [link]

The power of three-dimensional orientation detection of single emitting dipoles using a sophisticated scheme with three detectors in a confocal microscope is quantitatively explored by means of Monte Carlo simulations. We show that several hundreds of photons are sufficient for a reliable orientation determination. In typical single-molecule experiments, time resolutions in the submillisecond range for orientation trajectories become accessible. Experimental data on fluorescent latex beads and single perylene monoimide molecules show that a properly aligned setup can perfectly reproduce the simulated data. The simulations and experimental data highlight the potential of our method and give practical guidelines for its application.

Publication: Confined diffusion in ordered nanoporous alumina membranes

J. Hohlbein, M. Steinhart, C. Schiene-Fischer, A. Benda, M. Hof and C.G. Hübner, Small, 3, 380, 2007, [link]

Self-ordered nanoporous alumina was used to create a two-dimensional geometrical confinement for either single diffusing molecules or fluorescent polymerized nanowires. The membranes for measurements of single molecule diffusion featured a pore diameter of 35-40nm, a porosity (volume fraction of the pores) of 20-25%, and a thickness of 35 µm. Thus, the aspect ratio of the pores is ≃1000. In comparison, the size of the diffraction limited laser focus is roughly 2µm in height (long axis) and 0.5µm in diameter (short axes), resulting in an aspect ratio of 4. Therefore, if the long axis of the pores is aligned with the long axis of the confocal microscope, the probe molecules are forced to diffuse parallel to the long axis of the laser focus. Apparent one-dimensional diffusion within nanoporous alumina was shown for different probes such as Alexa Fluor 488 and the enhanced green fluorescent protein (eGFP). As compared to three-dimensional diffusion in free solution, the mean diffusion time through the focus increases within the pores. The factor of increase was theoretically derived as the squared aspect ratio of the laser focus resulting in a value of 16. Indeed, for Alexa Fluor 488 an increase in the mean diffusion time by a factor of 19 was found. In the case of eGFP a factor of 14 was obtained.