The free charge carrier lifetime is a highly sensitive parameter that can be used for analyzing the function of semiconductor devices and monitoring the quality of wafer materials. In this context, time-resolved photoluminescence (TRPL) is presented as a technique for directly determining the free charge carrier lifetime with pulsed diode laser excitation and time-correlated single photon counting, employing highly sensitive single-photon avalanche detectors (SPADs). The full range in time-scales of charge carrier dynamics can be addressed with the capability to resolve luminescence lifetimes from approximately 50 ps up to several hundred microseconds. This is achieved with an instrument response function (IRF) as short as 100 ps and the capability of adjustable repetition rates in the pulsed laser excitation that can be adapted to the luminescence lifetime of the material. The technique is capable of correlating spectral information concerning material specific band gap transitions and transmission edges with the respective luminescence lifetimes in a specific spectral channel. This is particularly valuable for the analysis of multi-component systems. Furthermore, the general instrumentation can be combined with a raster scanning based microscope setup, which can be configured to cover lateral resolutions down to sub-μm scale and scan ranges from 100 microns up to several centimeters [1].

Depending on the semiconductor material, the luminescence lifetime of semiconductor wafers can vary over a broad range from microseconds for Si-wafers down to sub-nanoseconds for III/V and II/VI based thin film or organic materials. The lifetime of a given wafer sample depends on the free charge carrier dynamics and can therefore be affected by several parameters. An important example is the influence of bulk or surface defects [1], thus the lifetime is a possible indicator for wafer quality. On dye-sensitized solar cells, lifetime measurements are also useful to characterize the energy transfer process from the sensitizer to the conduction band [2].We have developed a setup for time-resolved photoluminescence measurements (TRPL) based on pulsed diode lasers and time-correlated single photon counting (TCSPC) with highly sensitive single photon detectors. Depending on the detector type, the instrument response function (IRF) can be as short as 100 ps and the laser pulse rate can be adapted to the luminescence lifetime of the material. The resolvable lifetimes extend from approx. 50 ps up to several hundred microseconds. The electronics can also be integrated into a microscope based setup for imaging with a lateral resolution down to the sub-mu m range [3] as well as testing the lifetime behaviour at different injection levels. We will show measurement results of the system on an GaAsP-based Quantum Well.

Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the local environment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or reveal different conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standard laser scanning confocal microscopes towards a time-resolved system, which is based on picosecond pulsed lasers, fast detectors and sophisticated single photon counting electronics.We demonstrate the capabilities of the time-resolved approach by using fluorescence lifetime measurements to detect fluorescence resonance energy transfer (FRET) in living cells. The results show that different FRET-efficiencies can be spatially resolved within a single cell. Furthermore, the upgrade kit does not only allow to measure FRET by observing the shortening of the donor lifetime, but also the acceptor decay can be simultaneously monitored using two spectrally separated detectors and a router.A very special feature of the upgrade kit is that it uses an unrestricted data acquisition approach. With this approach, not only Fluorescence Lifetime Imaging Microscopy (FLIM) with single molecule sensitivity is realized, but the provided information can also be combined with other techniques such as Fluorescence Correlation Spectroscopy (FCS). This opens the way to complete new analysis and measurement schemes like Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to remove the influence of detector afterpulsing, which is classically done by cross correlation between two detectors.

We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 nm, 405 nm, 440 nm and 470 nm for fluorescence excitation which are coupled in through a sole single mode fiber. The detected signal is guided to a photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC5,7 system, via the unique Time-Tagged Time-Resolved (TTTR)6 data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently fit the full fluorescence lifetime image. The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows not only to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for the purpose of Fluorescence Lifetime Imaging (FLIM) or advanced Fluorescence Resonance Energy Transfer (FRET) analysis. We present here some selected results acquired with standard laser scanning microscopes upgraded for the time-correlated single photon counting technique.

This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in ''spot scan'' mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.

Fluorescence lifetime measurement of organic fluorophores is a powerful tool for distinguishing molecules of interest from background or other species. This is of interest in sensitive analysis and Single Molecule Detection (SMD). A demand in many applications is to provide 2-D imaging together with lifetime information. The method of choice is then Time-Correlated Single Photon Counting (TCSPC). We have devloped a compact system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner holding the immobilized sample. The system allows count rates up to 3 MHz and a resolution down to 30 ps. An overall Instrument Response Function down to 300ps is achieved with inexpensive detectors and diode lasers. The board is designed for the PCI bus, permitting high throughput without loss of counts. It is reconfigurable to operate in different modes. The Time-Tagged Time-Resolved (TTTR) mode permits the recording of all photon events with a real-time tag allowing data analysis with unlimited flexibility. We use the Time-Tag clock for an external piezo scanner that moves the sample. As the clock source is common for scanning and tagging, the individual photons can be matched to pixels. Demonstrating the capablities of the system we studied single molecule solutions. Lifetime imaging can be performed at high resolution with as few as 100 photons per pixel.

In single molecule detection by laser induced fluorescence, a main problem is the low signal to noise ratio due to scattering of the exciting laser light. One common approach to solve this problem is the application of time resolved techniques. Here we present a high speed electronic (based on a pair of PC cards) specially suited for detection of TCSPC curves in a continuous flow system. The whole system works on two different time scales: a millisecond time scale (every millisecond a complete TCSPC curve is measured and stored) and a picosecond time scale (showing the fluorescence decay). The technique present here is of particular interest for applications such as fast DNA sequencing, where a distinction between the different bases solely by the decay times of the attached fluorescence labels is conceivable.

Recently, major advances have been reached in the fluorescence detection of small amounts of molecules in liquids, making possible even the detection of single molecules in liquid flows. Significant improvements of fluorescence detection techniques make single molecule detection feasible for many applications, especially in the field of molecular biology and genetics. For such techniques new compact and inexpensive lasers are desirable. laser diode systems are the most favorable candidates for such light sources. With the expanding number of available NIR-fluorescent dyes, the importance of cheap and reliable laser light sources above 630 nm will increase. But not only cw-laser systems are of growing interest. In a number of recent papers, the application of time-resolved fluorescence detection down to a single molecule level was shown to be of great use for further improving detection efficiency. Thus, one needs high- repetition rate pulsed laser diode systems with good time and optical performance, and detection electronics with high-speed and large data throughput. Here we present such a system, combining a pulsed diode laser system with excellent electrical and optical parameters, and a high speed electronic for time correlated single photon counting. This system is suitable for a broad range of applications in ultra sensitive fluorescence detection.

We present data of simultaneously time and spectrally resolved measurements with the time correlated single photon counting technique using our new SPC-300 PC plug-in module combined with a 64 multianode PMT from Philips and a specially designed router. The SPC- 300 card records in up to 128 time channels simultaneously with a count rate of up to 5 MHz. In this paper we show the performance of the electronics for single decay curves using a MCP-PMT detector from Hamamatsu (R 3809U) and for wavelength resolved detection using an 8*8 multi-anode PMT from Philips (XP1702). An ultimate instrumental response function (IRF) of 34 ps and a maximum count rate of 300 000 cps was achieved by using the ultrafast MCP-PMT and a subtractive double monochromator. By using the multimode PMT coupled to a polychromator we got an IRF of 800 ps at 2.3*106 cps. The fluorescence signal was recorded at 8 different wavelengths simultaneously. For the test measurements we used pure Fluorescein, Rhodamin 6G and DODCI solution as well as a mixture of Fluorescein and DODCI. We got an excellent distinction between the two species. The decay times (3.9 ns, 1.1 ns) are in good agreement with the single curve measurement at a fixed wavelength.

Our new developed time-correlated single photon counting electronics allows for continuous registration of time-resolved fluorescence signals in millisecond intervals. With this electronics, adapted fluorescence measurement system, we studied single molecule transitions through a focused laser beam. In our paper we present measurements on single rhodamine 6G- molecules and discuss data processing methods, using the full information of the time resolved measurement. The use of this information influences significantly the reliability of the detection of single molecule transits. Further developments and applications of this method are discussed.

We will demonstrate the operation of the very compact all solid state fluorescence lifetime measurement system FLUO-TIME BQ 2759A. For this purpose we developed a new type of compact driving generator LD 4000 for a set of ps-laserdiodes with wavelengths between 630 nm and 690 nm, which will produce sub 50 ps pulses with up to 200 mW peak power and 3 MHz repetition rate. Using this miniaturized excitation source we are able to investigate a lot of red and NIR dyes. The fluorescence signal will be detected with single photon counting sensitivity by an ultrafast photomultiplier tube with only the size of the transistor (TO8 housing). Spectral resolution is given by a set of bandpass filters or a compact monochromator. With our recently introduced time correlated single photon counting (TCSPC) electronics SPC 300 (a PC-plug-in-card) we have a powerful instrument for data acquisition with highest data throughput. The instrumental response time (IRF) of the complete measurement system is less than 250 ps, allowing the investigation of fluorescence decay time components down to 25 ps using out deconvolution and analysis software package PHYSFIT. This performance can be improved to less than 90 ps IRF using a microchannel plate photomultiplier tube (MCP-PMT) detector. In this paper we demonstrate also the first practical application of this system to standard fluorescence dyes (oxazine, rhodamin).

In dichloromethane solutions were reacted [Rh(COD)Cl]2 (COD = cis,cis-1.5-cyclooctadiene) with each of the four new ligands of the type o-Py(CH2)2P(Ph)(CH2)3ZR in the presence of the halogen scavenger TlPF6 at 0-degrees-C to complex salts [Rh(COD)(o-Py(CH2)2P(Ph)(CH2)3ZR]PF6 (ZR = OC2H5, I; OPh, II; NHPh, III; NHcyclo-C6H11, IV). The Rh(I) complex cation in the obtained compounds I - IV coordinates besides the bidentate COD group the ligand donor atoms P und pyridinic N and the remaining donor atom Z is uncoodinated in an assumed square planar ligand geometry at the Rh central atom. In 1.4 dioxane solutions the complex catalysts I - IV polymerize at 25-degrees-C the substrate phenylacetylene (PA) to polyphenylacetylene (PPA): values of TON [h-1] between 352 (I) and 876 (IV), and average molecular weights M(w) (GPC measurements) between 238000 (I) and 199900 (IV). These given values exhibit a dependency on the ZR group in complexes I - IV. The microstructure of isolated PPA is cis-transoidal. It is formed stereospezific and, based on MNDO calculations, is thermodynamically favoured. For the purpose of comparison, from both the newly synthesized compounds of the type [Rh(COD)DBN- (or DBU)Cl] (DBN = 1.5-Diazabicyclo[4.3.0.]non-5-en, DBU = 1.8-Diazabicyclo[5.4.0]-undec-7-en) was obtained a larger value of TON with 1 292 (or 1 327) [h-1], but a lower value of M(w) with 166200 (or 131 200). These catalysts including I - IV polymerize PA to PPA at a lower reaction temperature with improved selectivity and larger values of M(w) as hitherto known catalyst systems.

AbstractIn Dichlormethanlösung wurde [Rh(COD)Cl]2 (COD = cis,cis‐1.5‐Cyclooctadien) mit vier neuen dreizähnigen Liganden vom Typ o‐Py(CH2)2P(Ph)(CH2)3ZR in Gegenwart des Halogenfängers TIPF6 bei 0°C zu Komplexsalzen [Rh(COD)(o‐Py(CH2)2P(Ph)(CH2)3ZR)] PF6 (ZR = OC2H5, I; OPh, II; NHPh, III; NHcycloC6H11, IV) umgesetzt. Das Rhodium‐Komplexkation in den abgetrennten Verbindungen I − IV koordiniert aufgrund von 1H−, 31P‐, 13C–NMR und IR–Meßergebnissen außer der zweizähnigen COD‐Gruppe die Donoratome Phosphor und Pyridinstickstoff und läßt in der angenommenen quadratisch‐planaren Koordinationsgeometrie ein vorhandenes Z‐Donoratom ungebunden. Die Komplexkatalysatoren I – IV polymerisieren in 1,4‐Dioxanlösung bei 25°C das Substrat Phenylacetylen (PA) zu Polyphenylacetylen (PPA): Werte für TON [h−1] von 352(I) und 876(IV) und mittlere Molmassen Mw (GPC‐Meßresultate) zwischen 238 000(I) und 199 900(IV). Diese Werte der Lösungs‐Fällungspolymerisation zeigen eine Abhängigkeit von der unkoordinierten ZR‐Gruppe in Komplexen I – IV. Die Mikrostruktur des isolierten PPA ist cis‐transoidal. Sie entsteht stereospezifisch und ist nach MNDO‐Rechnungen die thermodynamisch begünstigte. Von den zu Vergleichszwecken dargestellten neuen Rhodium(I)‐Komplexen des Typs [Rh(COD)DBN(bzw. DBU)Cl] (DBN = 1,5‐Diazabicyclo[4.3.0]‐non‐5‐en, DBU = 1,8‐Diazabicyclo[5.4.0]undec‐7‐en) wird unter analogen Reaktionsbedingungen für PA ein höherer TON‐Wert 1 292 (bzw. 1 327) [h−1], aber kleinerer Mw‐Wert von 166 200 (bzw. 131 200) erhalten. Gegenüber bisher bekannten Katalysatorsystemen polymerisieren diese Komplexkatalysatoren einschließlich I – IV PA zu PPA bei milderen Reaktionstemperaturen, verbesserter Selektivität und höheren Mw‐Werten.

Chloro(eta4-1,5-cyclooctadiene)(1,8-diazabicyclo[5.4.0]undec-7-ene)rhodium(I), (I), [RhCl(C8H12)(C9H16N2)], M(r) = 398.8, monoclinic, P2(1), a = 7.398 (1), b = 11.439 (2), c = 10.727 (2) angstrom, beta = 106.42 (1)-degrees, V = 870.7 angstrom3, Z = 2, D(x) = 1.521 Mg m-3, lambda(Mo Kalpha) = 0.71073 angstrom, mu = 1.12 mm-1, F(000) = 412, T = 296 (1) K, final R = 0.026, wR = 0.029 for 1912 unique observed intensities. Chloro(eta4-1,5-cyclooctadiene)(1,5-diazabicyclo[4.3.0]non-5-ene)rhodium(I) dioxane solvate, (II), [RhCl(C8H12)(C7H12N2)].1/2C4H8O2, M(r) = 414.8, monoclinic, P2(1)/c, a = 7.231 (1), b = 15.870 (3), c = 15.918 (3) angstrom, beta = 100.40 (1)-degrees, V = 1796.7 angstrom3, Z = 4, D(x) = 1.533 Mg m-3, lambda(Mo Kalpha) = 0.71073 angstrom, mu = 1.09 mm-1, F(000) = 856, T = 296 (1) K, final R = 0.044, wR = 0.048 for 3203 unique observed intensities. In the two complexes each Rh atom is coordinated by one Cl, two double bonds of cod and one N of the corresponding ligand realizing a slightly distorted square planar geometry. Substitution of dbu vs dbn leaves the rhodium-ligand bond lengths nearly unchanged, the deviation from planarity, however, being significantly greater for the dbu complex.

Several iron‐containing fertilizers such as FeSO4 Fe‐EDTA, Fe‐EDTA + urea, Fe‐EDTA + superphosphate and a “commercial product”; (SD 807) containing N, Fe, Zn and Mn were applied to iron‐chlorotic soybean leaves and the effects on regreening and reorganization of iron‐chlorotic chloroplasts were studied. Fe‐EDTA + urea seemed most effective, but was in higher concentrations most likely to cause foliar injury. SD 807 had caused a lighter green than the other fertilizers. Reduction of grana‐stacking is characteristic of iron‐deficient chloroplasts. Concomitant with regreening, grana‐stacking of chloroplasts increased. Six days after foliar application the recovered chloroplasts resembled chloroplasts of control plants, although they contained fewer thylakoids per granum than control chloroplasts. Iron resupply to the nutrient solution, however, resulted in well‐developed chloroplasts, comparable with control chloroplasts. In more pronounced iron deficiency, when leaf colour appeared to be yellow‐white, chloroplasts displayed severe disintegration. In such cases it was not possible to achieve regreening by foliar fertilization. Key words: Iron deficiencyFoliar applicationSoybeanChloroplast ultrastructure

Iron chlorosis is a world-wide problem. Soils in which iron chlorosis occurs are referred to as high-lime, alkaline or calcareous. Iron chlorosis is also associated with high density of the soil, poor drainage after irrigation or with wet and cold weather conditions. Under these conditions iron is not available for continuous supply to the plant. As iron supplied to the soil is fixed under high pH values spray application of iron is the most common treatment in the field to correct iron deficiency chlorosis.