Danny Kowerko

Formation Principles and Ligand Dynamics of Nanoassemblies of CdSe Quantum Dots and Functionalised Dye Molecules

Functional dye molecules, such as porphyrins, attached to CdSe quantum dots (QDs) through anchoring meso-pyridyl substituents, form quasi-stable nanoassemblies. This fact results in photoluminescence (PL) quenching of the QDs both due to Förster resonance energy transfer (FRET) and the formation of non-radiative surface states under conditions of quantum confinement (non-FRET). The formation process is in competition with the ligand dynamics. At least two timescales are found for the formation of the assemblies: 1) one faster than 60 s attributed to saturation of empty attachment sites and 2) one slower than 600 s, which is attributed to a reorganisation of the tri-n-octylphosphine oxide (TOPO) ligand shell. Finally, this process results in almost complete exchange of the TOPO shell by porphyrin dye molecules. Following a Stern-Volmer analysis, we established a microscopic description of PL quenching and assembly formation. Based on this formalism, we determined the equilibrium constant for assembly formation between QDs and the pyridyl-functionalised dye molecules to be K ≈ 10(5) - 10(7)  M(-1), which is several orders of magnitude larger than that of the TOPO ligands. Our results give additional insights into the non-FRET PL quenching processes involved and show that the QD surface is inhomogeneous with respect to the involved attachment and detachment processes. In comparison with other methods, such as NMR spectroscopy, the advantage of our approach is that ligand dynamics can be investigated at extremely low ratios of dye molecules to QDs.

Identification of Different Donor-Acceptor Structures via Förster Resonance Energy Transfer (FRET) in Quantum-Dot-Perylene Bisimide Assemblies

Nanoassemblies are formed via self-assembly of ZnS capped CdSe quantum dots (QD) and perylene bisimide (PBI) dyes. Upon assembly formation the QD photoluminescence is quenched, as can be detected both via single particle detection and ensemble experiments in solution. Quenching has been assigned to FRET and NON-FRET processes. Analysis of FRET allows for a distinction between different geometries of the QD dye assemblies. Time-resolved single molecule spectroscopy reveals intrinsic fluctuations of the PBI fluorescence lifetime and spectrum, caused by rearrangement of the phenoxy side groups. The distribution of such molecular conformations and their changed dynamics upon assembly formation are discussed in the scope of FRET efficiency and surface ligand density.

Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics.

We report on the simultaneous detection of the fluorescence lifetime, spectrum, and three-dimensional dipole orientation determination of single perylene diimide molecules deposited on a silica surface as a model system for studying fluorophore internal and orientational dynamics. We employ a multi-parameter detection scheme to demonstrate how jumps in the orientation of the molecule can be disentangled from spectral jumps, both leading to changes of the detected total fluorescence intensity. The fluorescence lifetime determined simultaneously from the same photons is also sensitive to the orientation of the dipole with respect to the interface between media with different refractive indices. The correlated changes of the lifetime and orientation we observe are in good agreement with theory.

Restricted conformation dynamics of single functionalized perylene bisimide molecules on SiO2 surfaces and in thin polymer films

Intramolecular conformational dynamics caused by the bay groups of perylene bisimide (PBI) molecules have been investigated by single molecule spectroscopy in thin PMMA polymer films and on SiO2 surfaces. All dynamic processes occur in a jump-like fashion on SiO2 but in a likewise continuous way in PMMA. Surface attachment on SiO2 is accomplished by pyridyl functionalization of PBI demonstrating a nearly perpendicular orientation of the long axis of perylene as has been concluded from polarization experiments. This orientation is subject to fluctuations of the molecular orientation and/or conformation, which are considerably reduced in PMMA. According to static and time-dependent fluorescence spectra at least 2 different conformations have been identified.

Cation-induced kinetic heterogeneity of the intron–exon recognition in single group II introns

Significance RNAs are involved in numerous aspects of cellular metabolism, and correct folding is crucial for their functionality. Folding of single RNA molecules can be followed by single-molecule spectroscopy. Surprisingly, it has been found that chemically identical RNA molecules do often not behave identically. The molecular origin of this heterogeneity is difficult to rationalize and the subject of ongoing debate. By combining single-molecule spectroscopy with NMR, we show that heterogeneity is likely to stem from a subset of microscopically different RNA structures that differ with regard to the occupation of divalent metal ion binding sites. RNA is commonly believed to undergo a number of sequential folding steps before reaching its functional fold, i.e., the global minimum in the free energy landscape. However, there is accumulating evidence that several functional conformations are often in coexistence, corresponding to multiple (local) minima in the folding landscape. Here we use the 5′-exon–intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg2+ and Ca2+ on RNA tertiary structure formation. Bulk and single-molecule spectroscopy reveal that near-physiological M2+ concentrations strongly promote interstrand association. Moreover, the presence of M2+ leads to pronounced kinetic heterogeneity, suggesting the coexistence of multiple docked and undocked RNA conformations. Heterogeneity is found to decrease at saturating M2+ concentrations. Using NMR, we locate specific Mg2+ binding pockets and quantify their affinity toward Mg2+. Mg2+ pulse experiments show that M2+ exchange occurs on the timescale of seconds. This unprecedented combination of NMR and single-molecule Förster resonance energy transfer demonstrates for the first time to our knowledge that a rugged free energy landscape coincides with incomplete occupation of specific M2+ binding sites at near-physiological M2+ concentrations. Unconventional kinetics in nucleic acid folding frequently encountered in single-molecule experiments are therefore likely to originate from a spectrum of conformations that differ in the occupation of M2+ binding sites.

OphthalVis: making data analytics of optical coherence tomography reproducible

In this paper, we discuss the issues of the current state of the art in optical coherence tomography with respect to reproducibility. We present our findings about the internal computations and data storage methods of the currently used devices. The gained knowledge was used to implement a tool to read a variety of OCT file formats and reproduce the visualizations used in daily clinical routine.

Time resolved 3D orientation spectroscopy: experimental realization and simulation

Confocal microscopy is a powerful tool for single molecule investigation of fluorescent macromolecules. Besides the commonly studied features in single molecule detection, the 3D orientation determination of the emission dipole enables the analysis of different conformational states. These conformational states can be represented as state depending dipole orientations intrinsic to the fluorescent molecule and/or in relation to the molecular frame. They might be subject to intramolecular dynamics, which may lead to spectral diffusion, fluorescence intensity and/or lifetime fluctuations and changes in the orientation of the emission dipole. We demonstrate a detection scheme that allows for simultaneous determination of the full 3D emission dipole orientation, the fluorescence intensity, the fluorescence lifetime and the emission spectra of single fluorescent molecules. We evaluate the feasibility of our approach using pyridyl functionalized perylene bisimide (PBI) as a model system exhibiting conformational changes. Moreover, MC simulations demonstrate the full potential of our detection scheme to distinguish between intensity fluctuations due to conformational changes and changes in the out-of-plane orientation or changes in both of them.

Detailed analysis of complex single molecule FRET data with the software MASH

The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).

Simulations of camera-based single-molecule fluorescence experiments

Single-molecule microscopy has become a widely used technique in (bio)physics and (bio)chemistry. A popular implementation is single-molecule Förster Resonance Energy Transfer (smFRET), for which total internal reflection fluorescence microscopy is frequently combined with camera-based detection of surface-immobilized molecules. Camera-based smFRET experiments generate large and complex datasets and several methods for video processing and analysis have been reported. As these algorithms often address similar aspects in video analysis, there is a growing need for standardized comparison. Here, we present a Matlab-based software (MASH-FRET) that allows for the simulation of camera-based smFRET videos, yielding standardized data sets suitable for benchmarking video processing algorithms. The software permits to vary parameters that are relevant in cameras-based smFRET, such as video quality, and the properties of the system under study. Experimental noise is modeled taking into account photon statistics and camera noise. Finally, we survey how video test sets should be designed to evaluate currently available data analysis strategies in camera-based sm fluorescence experiments. We complement our study by pre-optimizing and evaluating spot detection algorithms using our simulated video test sets.

Camera-based color measurement of DLP projectors using a semi-synchronized projector camera system

Modern DLP projectors often use a time-multiplex approach to generate color: A rotating color wheel is used to project the red, green and blue components (and possibly more) as separate sub-images which are each displayed for a short period of time. Applications like color calibration require high quality measurements of the color output of the projector, which might be acquired with digital cameras. When capturing the output of a DLP projector with a color wheel, the timing of the projector in relation to the exposure time must be taken into account, to avoid deviations introduced by capturing fractional parts of a color wheel rotation. In this work, we demonstrate the feasibility of software-only semi-synchronization between a DSLR camera and a DLP projector, using only a PC, camera with an USB interface and a projector connected via HDMI. We found that a reasonable estimate of the end of the actual exposure can be acquired with millisecond precision. By relating that to the previous vertical blanking interval, we are able to reconstruct the position of the color wheel throughout the exposure. Using a multitude of photos, it is possible to measure the actual color wheel timing of the projector for a specified input color. Furthermore, we show that this data can be used to build a simple model of the image formation process of the projector, which enables the compensation of color deviations introduced by the incomplete rotation. We show that using our compensation technique significantly improves the accuracy of the color measurements for reasonable exposure times.