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Purpose: Despite numerous studies demonstrating promising short-term outcomes of Total Knee Arthroplasty (TKA) with Customized Individually Made (CIM) implants, there is a significant lack of data on their mid-term effectiveness. Given the increasing number of TKAs performed annually, the rising demand for CIM implants, and the associated burden of revision surgeries, understanding the mid-term performance of CIM implants is crucial. Therefore, this study aims to report on the mid-term (minimum 5 years) outcomes of TKA using a CIM implant., Methods: This retrospective cohort study included a consecutive series of 116 patients who received the ConforMIS ® iTotal CR implant between 2015 and 2018. Inclusion criteria were end-stage knee osteoarthritis with coronal deformities below 10° and absence of ligamentous instability. Exclusion criteria included simultaneous bilateral TKA. Patients were followed up at a minimum of five years post-surgery. They completed a questionnaire reporting on satisfaction, pain levels using the Visual Analogue Scale (VAS), current weight, the Oxford Knee Score (OKS), and the Forgotten Joint Score for the knee (FJS-knee). Statistical analysis included descriptive statistics for demographic and clinical variables, and outcomes were reported as means with ranges., Results: The mean follow-up duration was 5.9 ± 0.8 years (range 5-7.4 years). 90% of patients stated they would undergo the same operation again, and 93% were either satisfied or very satisfied. The mean VAS for pain at rest was 2 ± 1.5 (range 0-6) and during exercise was 3 ± 2 (range 0-8). 58 patients (53%) managed to lose weight. The mean OKS was 41 ± 9 points (range 15-48), and the mean FJS-knee was 67 ± 23 points (range 4-100). No severe complications occurred., Conclusion: CIM TKA using the ConforMIS ® iTotal CR implant can achieve excellent results with 93% of patients being satisfied or very satisfied at mid-term follow-up of five years. Prospective, randomized, and patient-blinded trials comparing off-the-shelf (OTS) TKAs with CIM implants are necessary to evaluate whether these implants are superior or not., (© 2024. The Author(s).)

Background : Bucket-handle meniscal tears are mostly treated arthroscopically. However, there is no clear evidence as to whether the time to surgery impacts the outcome and whether or not lesions should be treated urgently. Methods : Sixty patients were interviewed about the current status of their knee with a mean follow-up of 6.1 years (SD = 3.5). Forty-one patients underwent meniscus repair, and fifteen patients received partial resections. The primary outcome was the rate of reoperation after meniscus repair. Secondary outcomes were pain at rest and during exercise, return to sports, and Tegner and Lysholm scores. Results : The average time to surgery was 14.4 days, with no significant impact of surgical timing on the rate of reoperation. Furthermore, no significant differences were found in pain levels, return to sports, or Tegner and Lysholm scores based on the timing of surgery. Conclusions : In our cohort, the time to surgery was not a prognostic factor for the reoperation rate or postoperative outcome in repairing bucket-handle meniscal tears. Therefore, arthroscopic repair should not be performed in an emergency setting but conducted after careful planning by experienced arthroscopy surgeons. Regarding the return to sports, postoperative factors such as rehabilitation protocols or surgical techniques could be more important than the time to surgery.

The optimal ventilation strategy during cardiopulmonary resuscitation (CPR) has eluded scientists for years. This porcine study aims to validate the hypothesis that ultra-low tidal volume ventilation (tidal volume 2-3 mL kg -1 ; ULTVV) minimizes renal and hepatic end-organ damage when compared to standard intermittent positive pressure ventilation (tidal volume 8-10 mL kg -1 ; IPPV) during CPR. After induced ventricular fibrillation, the animals were ventilated using an established CPR protocol. Upon return of spontaneous circulation (ROSC), the follow-up was 20 h. After sacrifice, kidney and liver samples were harvested and analyzed histopathologically using an Endothelial, Glomerular, Tubular, and Interstitial (EGTI) scoring system for the kidney and a newly developed scoring system for the liver. Of 69 animals, 5 in the IPPV group and 6 in the ULTVV group achieved sustained ROSC and were enlisted, while 4 served as the sham group. Creatinine clearance was significantly lower in the IPPV-group than in the sham group ( p < 0.001). The total EGTI score was significantly higher for ULTVV than for the sham group ( p = 0.038). Aminotransferase levels and liver score showed no significant difference between the intervention groups. ULTVV may be advantageous when compared to standard ventilation during CPR in the short-term ROSC follow-up period.

This report highlights the combination of the MicroTime 100 upright confocal fluorescence lifetime microscope with a Single Quantum Eos Superconducting Nanowire Single-Photon Detector (SNSPD) system as a powerful tool for photophysical research and applications. We focus on an application in materials science, photoluminescence imaging, and lifetime characterization of Cu(InGa)Se 2 (CIGS) devices intended for solar cells. We demonstrate improved sensitivity, signal-to-noise ratio, and time-resolution in combination with confocal spatial resolution in the near-infrared (NIR) range, specifically in the 1000-1300 nm range. The MicroTime 100-Single Quantum Eos system shows two orders of magnitude higher signal-to-noise ratio for CIGS devices' photoluminescence imaging compared to a standard NIR-photomultiplier tube (NIR-PMT) and a three-fold improvement in time resolution, which is now limited by the laser pulse width. Our results demonstrate the advantages in terms of image quality and time resolution of SNSPDs technology for imaging in materials science.

This report highlights the combination of the FluoTime 300 photoluminescence spectrometer with a FluoMic add-on as a powerful tool for photophysical research and applications, yielding spectral, temporal, and spatial information on a wide range of samples. The steady-state and time-resolved measurement capabilities of this combination are demonstrated reflecting a broad range of applications.

Objective: A novel high-precision approach [lifetime-decomposition measurement (LTDM)] for the assessment of the glomerular filtration rate (GFR) based on clearance measurements of exogenous filtration marker., Methods: The time-correlated single photon counting (TCSPC) acquisition in combination with a new decomposition method allows the separation of signal and background from transcutaneous measurements of GFR., Results: The performance of LTDM is compared versus the commercially available NIC-kidney patch-based system for transcutaneous GFR measurement. Measurements are performed in awake Sprague Dawley (SD) rats. Using the standard concentration required for the NIC-kidney system [7-mg/100-g body weight (b.w.) FITC-Sinistrin] as reference, the mean difference (bias) of the elimination curves GFR between LTDM and NIC-kidney was 4.8%. On the same animal and same day, the capability of LTDM to measure GFR with a FITC-Sinistrin dose reduced by a factor of 200 (35-μg/100-g b.w.) was tested as well. The mean differences (half lives with low dose using LTDM compared with those using first, the NIC-Kidney system and its standard concentration, and second, LTDM with the same concentration as for the NIC-Kidney system) were 3.4% and 4.5%, respectively., Conclusion: We demonstrate that with the LTDM strategy substantial reductions in marker concentrations are possible at the same level of accuracy., Significance: LTDM aims to resolve the issue of the currently necessary large doses of fluorescence tracer required for transcutaneous GFR measurement. Due to substantially less influences from autofluorescence and artifacts, the proposed method outperforms other existing techniques for accurate percutaneous organ function measurement.

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes.

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes., (© 2014 John Wiley & Sons Ltd.)

Background/aims: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens., Methods: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures., Results: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes., Conclusion: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments., (Copyright © 2011 S. Karger AG, Basel.)

Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico- to nano-molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non-ideal confocal volume can be experimentally determined and validated.

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2-U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 microm2/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.

During the last years, Fluorescence Correlation Spectroscopy (FCS) has proven to be a powerful tool for basic research in many applications. The combination of a minimal detection volume in the femtoliter range coupled with very high sensitivity extends the possibilities to design sensitive homogeneous tests. In this article we illustrate the analysis of binding processes with FCS based on the changes in diffusion characteristics of GFP upon binding to an antibody. Problems induced by highly heterogeneous samples are discussed and differences of GFP binding to a monoclonal and a polyclonal antibody are shown and analyzed. We stress data processing, limitations and useful approximations in FCS methodology. Basic ideas of data acquisition and processing as well as new developments and applications are presented.

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.

In this report, a combined imaging and fluorescence correlation spectroscopy (FCS) method is described and its ability to characterize microsecond fluctuations in the fluorescence emission of a sample is demonstrated. A sample scanning laser confocal microscope is operated in the customary way while recording the time that each photon is detected with a time resolution of 50 ns using a low-cost counting board. The serial data stream of photon detection times allows access to fluorescence signal fluctuations that can be used to characterize dynamics using correlation methods. The same data stream is used to generate images of the sample. Using the technique, we demonstrate that it is possible to characterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC16 and ATTO 520. Results are similar to, but deviate slightly from, a model that has been frequently used for extracting singlet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration, or in our case the areal density of coverage, of fluorescent species can also be obtained. Many single-molecule fluorescence experiments aim to extract kinetics from intensity trajectories; this method may be used as a rapid and convenient technique for characterization of surface-linked or thin-film samples prior to performing the more time and effort intensive single-molecule measurements. Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also be applied for measuring conformational changes or interaction kinetics for species immobilized on a surface. One possible scenario is measurements of the frequency and duration of association of ligand-receptor pairs where a fluorescently labeled component is freely diffusing and the other is surface immobilized. Given that microarrays of custom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of the surface-linked species with a freely diffusing species could be a useful extension to what has already become an extremely important tool for characterizing the interactions of biomolecules.

In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micrometer resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy (FCS). Flow velocities ranging from approximately 50 microm/s to approximately 10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion tracers. Velocity profiles and images of the flow in poly(dimethylsiloxane)-glass microchannels are presented and analyzed. Using the method, velocity images along the horizontal (top view) and vertical planes within a microdevice can be obtained. This is, to our knowledge, the first report of FCS for producing velocity maps. The high-resolution velocity maps can be used to characterize and optimize microdevice performance and to validate simulation efforts.

Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various [H-258]/[DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a [H-258]/[DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of approximately 3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low [H-258]/[DNA bp] ratios. With increasing [H-258]/[DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher [H-258]/[DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher [H-258]/[DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher [H-258]/[DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of approximately 500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.

Tetrapropylammonium (TPA)-containing precursors are the building blocks in the crystallization of silica. In the first steps slab-shaped silicalite nanoparticles are formed by ordered combination of the precursors. These nanoslabs have MFI-type zeolite framework topology and play a key role in TPA-ion-mediated zeolite crystallization from monomeric and polymeric silica sources., (© 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.