Sebastian Tannert

Electron donor–acceptor compounds: exploiting the triptycene geometry for the synthesis of porphyrin quinone diads, triads, and a tetrad

Abstract Rigidly and covalently linked porphyrin quinones are well established as model compounds for studying photo-induced electron transfer (PET) reactions like those occurring during the primary processes of photosynthesis. In this context, the synthesis of a number of porphyrin quinones is reported in which one or two porphyrin electron donors are connected to either one or two quinone electron acceptors, resulting in diad, triad, and tetrad model systems, respectively. The porphyrin(s) and the quinone(s) are linked by triptycene, 1,4-phenylene, and cis - or trans -1,4-cyclohexylene bridges. The use of the 1,4-phenylene, and cis - or trans -1,4-cyclohexylene bridges results in donor–acceptor compounds with the same number of bonds between donor and acceptor(s), but differing in distance and orientation. Analysis of the 1 H NMR spectra confirmed the chair conformation for the cis - and trans -cyclohexylene-linked diads and triads. NOE experiments gave information about the spatial arrangement of the target compounds. The key compounds in the syntheses of all these new PET model systems are the triptycene quinones, which are formed via a [π s 4 +π s 2 ]-cycloaddition between an anthracene derivative and a suitable quinone. The triptycene system enforces a rigid orientation on the quinone acceptor(s) in the final model system. Evidence is given that the triptycene system has further potential for constructing tailor-made donor–acceptor compounds.

Annulated Dinuclear Metal-Free and Zn(II) Phthalocyanines : Photophysical Studies and Quantum Mechanical Calculations

The results of steady-state and time-resolved absorption and fluorescence experiments as well as quantum mechanical density functional theory (DFT) calculations of metal-free and Zn(II) mononuclear and dinuclear (sharing a common benzene ring) phthalocyanines are presented. A detailed comparison between measured and calculated absorption spectra of all compounds is done, showing a good agreement between theory and experiment. The NH tautomerization for phthalocyanines with an extended pi-electron system was shown for the first time at room temperature. The photophysical properties of all possible NH tautomers of metal-free dinuclear Pc have been fully characterized. In the first tautomer, Pc(parallel), both pairs of hydrogen atoms are parallel to the connection line of two Pc units. The maximum of the lowest-energy Q absorption band, lambda abs, in Pc(parallel) is located at 832 nm, whereas the spectral position of the fluorescence maximum lies at lambdafl=837 nm. The second NH tautomer, Pc(perpendicular) (lambdaabs=853 nm, lambdafl=860 nm), presents the two pairs of hydrogen atoms perpendicularly orientated to the covalent axis, and the third one, Pc(mix) (lambdaabs=864 nm, lambdafl=872 nm), contributing in a minor extend to the absorption and fluorescence spectra of the metal-free dinuclear phthalocyanine, has one perpendicular and one parallel pair of hydrogen atoms. Obviously, only one configuration exists in the case of the Zn(II)-containing dinuclear phthalocyanine (lambdaabs=845 nm, lambdafl=852 nm).

Optimizing a Time-Resolved Spectrometer for All Time Scales

Time-resolved fluorescence spectroscopy is a spectroscopists most valuable tool for the investigation of excited state dynamics in molecules, complexes, or semi-conductors. In recent years, the study of luminescence properties has gained in popularity in many scientific fields, including Chemistry, Biology, Physics, as well as in Life, Material or Environmental Sciences. The investigations to be carried out in each of these fields impose different requirements. On one side, monitoring dynamic processes in the excited state necessitates high time resolution that can be achieved by fast pulsed lasers and detectors along with appropriate time-correlated single photon counting (TCSPC) units and small monochromators. On the other hand, high spectral resolution is desirable for fluorophore characterization, requiring detectors with high quantum efficiencies, flash lamps for phosphorescence measurements and large monochromators. Up to now, spectrometers have been usually targeted towards either one of these two specifications. Spectrometers equipped with hybrid detectors, versatile TCSPC cards with optional longer time ranges, and pulsed lasers capable of working in a burst mode can offer an combined solution, covering most of the demands of either high time or spectral resolution. We will demonstrate the performance of such a spectrometer in terms of its time resolution, the ability to measure long decays and record time-gated spectra using laser drivers with burst capabilities. This type of instrument is of great value for analytical facilities in research centers, as it offers a wide range of possible spectroscopic applications in a single, easy to use instrument.

Multispecies imaging and diffusion studies using super-resolution microscopy

Cell membranes play an active role in many biological processes and are complex, highly heterogeneous media. They contain a variety of molecular species and structural elements that may interact with each other, leading to complex lateral diffusion behavior. Insights into this behavior could help in understanding the composition, organization, and function of membranes in cell biology. Fluorescence-correlation spectroscopy (FCS) is a powerful method for determining the average diffusion coefficients of the molecules in these membranes. Measurements that are made using this approach are based on the movement of thousands of molecular transitions through an observation spot.1 However, the diffraction-limited resolution of confocal microscopy makes it difficult to probe heterogeneity on the sub-100nm scale. Stimulated-emission-depletion (STED) microscopy has become a well-established method for achieving spatial super resolution (below 50nm). Using STED enables the size of the observation volume to be tuned. As a result, the common issue that arises from fluorescence-correlation spectroscopy, namely, the diffraction-limited lateral resolution, is avoided. This limited resolution makes it difficult to probe diffusion heterogeneity on the sub-100nm scale and is only inferable by extrapolations. By gradually shrinking the observation spot size, the type of hindered diffusion (e.g., unperturbed Brownian motion, obstacleinduced restricted diffusion, or transient trapping of molecules in permeable domains), and therefore the local environmental organization of the diffusors, can be determined. Figure 1. Schematic overview of the time-resolved confocal microscope setup. The excitation and stimulated-emission-depletion (STED) lasers (640 and 765nm, respectively) are spatially overlaid and coupled into a single-mode fiber. Both beams pass through a quarter-wave plate, the main dichroic mirror, and the segmented phase plate. The beams are then focused on the sample with a high-numerical-aperture objective. Emitted light is collected and imaged through a pinhole onto a singlephoton avalanche diode (SPAD). The inset shows the back-reflection images of an 80nm gold bead for the excitation and STED lasers.

Advanced Pulse Pattern Generation and Fine Tuning for STED Microscopy

Stimulated Emission Depletion (STED) microscopy has evolved into an established imaging method offering super-resolution well beyond 50 nm. Whereas STED is now available in many laboratories, it is still in the focus of research to push the boundaries of its capabilities and applications. Time-resolved STED microscopy using time correlated single photon counting (TCSPC), is advantageous for many applications and promises further development for increased resolution and less photo-damage.Here, we show the application of established methods (e.g. gSTED) as well as emerging applications of time-resolved STED. We employ pulsed interleaved excitation (PIE), where the STED laser is pulsed at half the frequency of the excitation laser, such that STED and confocal data is taken practically at the same time. By using this approach, single molecule STED experiments can be carried out while the confocal control-experiment is performed simultaneously, allowing to account for measurement artifacts due to the high power of the STED laser. We will show examples from single molecule imaging, where blinking and bleaching are monitored using the confocal data. Furthermore, we will present STED-FCS data, where the confocal data allows insight into changes of the sample due to the STED laser. Since the control experiment for the influence of the STED laser is performed at the same time as the STED measurement, experimental parameters can be adjusted online to give highest resolution while ascertaining that the relevant information drawn from the experiment is not affected.Furthermore, we will present how electronically delaying the STED laser with respect to the excitation laser can increase resolution with no increase in photo-bleaching. By setting the arrival of the STED laser with an accuracy of about 20 ps, experimental conditions for fluorophores with different fluorescence lifetimes can be adjusted.

Sensitive Time-Correlated Single Photon Counting Enables Efficient Singlet Oxygen Detection

Single photon counting based data acquisition has proven to yield a major sensitivity increase in the optical evaluation of pharmaceuticals and biotechnology products. We will show for the first time that a state of the art time-correlated single photon counting (TCSPC) based fluorescence lifetime spectrometer is able to quantify singlet oxygen generation and to characterize the singlet oxygen phosphorescence decay. This makes TCSPC based fluorescence lifetime spectrometers a valuable tool for studying photosensitizers widely used for example in photodynamic therapy (PDT). The detection of the faint singlet oxygen phosphorescence signal has been made possible by using a special burst mode for the pulsed laser excitation and a new generation of TCSPC electronics with a significantly reduced dead-time which enables efficient multi-stop photon detection.Thanks to a recently developed integrating sphere add-on we are also able to measure fluorescence quantum yields with the same instrument. Leveraging the possibility to measure fluorescence lifetime in conjunction with quantum yield, we performed a systematic investigation of the relation between reduced fluorescence emission and different contributions of dynamic and static quenching processes.Furthermore, since quenching normally does not affect the radiative rate constant, this combined set-up allows to verify the accuracy of the extracted lifetime. Especially for very short lifetimes in the range of the instrument response function the presented method allows to assess whether the proper fluorescence lifetime was extracted.

Fast and Reliable Measurement of Photoluminescence Quantum Yields for the Development of Fluorescent Probes

Besides molar absorption coefficient and excited state lifetime, the photoluminescence quantum yield is the key parameter to be optimized for highly efficient and bright fluorescent probes. An easy and reliable way to determine the quantum yield is therefore the prerequisite for modern fluorescent probe development.Photoluminescence quantum yield measurements are typically done by comparing the emission intensity of the target compound with a standard of known quantum yield, under identical measurement conditions. This method is well established and precise, but also time consuming. In addition target compound and reference have to have similar absorption and emission spectra. For those cases where a suitable standard is not available; when the measurement of the absorption is cumbersome, when the determination speed is an issue, or generally for scattering samples, the use of an integrating sphere to measure the absolute photoluminescence quantum yield is mandatory.Here we show that absolute photoluminescence quantum yield measurements of solutions as well as solid samples can be easily realized using a simple integrating sphere accessory for a conventional fluorescence lifetime spectrometer. This allows to acquire all relevant fluorescence characteristics with one instrument, therefore streamlining the characterization workflow and keeping all calibration schemes simple.For the validation of our new assembly, selected quantum yield standards have been measured and the data were compared to literature data previously determined with a calibrated spectrofluorometer and two calibrated integrating sphere setups [1]. Procedures for the determination of the instruments spectral sensitivity and attainable precision of the results will be discussed.[1] Wurth, C.; Pauli, J.; Lochmann, C.; Spieles, M.; Resch-Genger, U. Anal. Chem. 2012, 84, 1345-1352.