Juergen M. Wolfrum

SIMULTANEOUS DIODE-LASER-BASED IN SITU DETECTION OF MULTIPLE SPECIES AND TEMPERATURE IN A GAS-FIRED POWER PLANT

We have developed a diode-laser (DL)-based spectrometer and demonstrated, to our knowledge, the first simultaneous in situ detection of all major combustion species and the temperature in the same measurement volume for active combustion control purposes and to ensure a safe ignition procedure of large-scale multi-burner gas-fired combustion systems. Two distributed-feedback DLs at 760 nm and 1.65 μ m were used to detect O 2 , CH 4 , and CO 2 , while a Fabry-Perot DL at 812 nm served to extract absolute H 2 O concentrations and the temperature from multiline water spectra. Permanent alignment of the laser beams could be ensured, despite strong wall deformation, with a new active alignment control loop. We analyzed the instationary ignition procedure of a full-scale gas-fired power plant with a 10 m furnace diameter using the spectrometer. A time resolution of 1.6 s and a minimum detectable absorption better than 10 −3 OD could be achieved. CH 4 could be detected with a dynamic range of more than two orders of magnitude and a detectivity in the 100 ppm range. A strong dependence of the CH 4 signal on the burner height was found. This spectrometer is well suited to enable an on-line control of the furnace atmosphere and a rapid detection of ignition delays by unburned CH 4 .

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

Luminescence and transient absorption bands in fused SiO2 induced by KrF laser radiation at various temperatures

Abstract High purity fused silica with about 800 ppm OH content was irradiated with KrF laser radiation (248 nm) at temperatures between −180°C and room temperature. The laser-induced absorption at 215 nm (due to E′ centers) was investigated. The generation rate of E′ centers from a precursor state depends linearly on the applied energy density, indicating the presence of a one-photon generation process for E′ centers in addition to the two-photon generation process from the silica network. The generation rate and relaxation of the 215 nm band is reduced at temperatures below room temperature. A luminescence band centered at 650 nm is excited during irradiation. A transient non-bridging oxygen center is postulated to explain the observed increase of the luminescence intensity with decreasing temperatures and with increasing radiation energy density.

Simultaneous antigen detection using multiplex dyes

To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.

Use of diode lasers in an evanescent wave immunosensor for stationary and time-resolved detection of antigens

Abstract A compact and inexpensive fiber-optic evanescent wave immunosensor is presented which uses a diode laser as excitation source both in continuous wave measurements as well as in time-resolved experiments. The continuous wave laser operates at 657 nm utilizing a commercially available dye-conjugated polyclonal antibody. Time-resolved experiments with a diode laser emitting at 674 nm were carried out with two fluorescent dyes, which have nearly identical absorption and emission wavelengths, but differ in their fluorescence lifetimes. This shows the possibility of detecting several molecules simultaneously, which are tagged with so-called ‘multiplex dyes’, by identifying the different fluorescence lifetimes. Using a new rhodamine dye a successful test of observing the time-resolved binding process between immobilized antibodies and dye-conjugated antigens is reported.

Diode laser based time-resolved detection and identification of individual mononucleotide molecules in aqueous solution

We applied a short-pulse diode laser emitting at 637 nm with a repetition rate of 30 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual labeled mononucleotide molecules in water. A newly synthesized oxazine dye and the commercially available carbocyanine dye Cy5 were used as fluorescent labels. Multichannel scalar traces, the fluorescence autocorrelation function and fluorescence decay times determined by time- correlated single-photon counting have been measured simultaneously. The time-resolved signals of the two mononucleotides were analyzed and identified by a maximum likelihood estimator. The results showed out that 60 detected photons per transit of a single molecule are sufficient to distinguish two labeled mononucleotides in water with a misclassification of less than 10 percent via their characteristic fluorescence lifetimes of 1.07 +/- 0.27 ns and 1.89 +/- 0.34 ns.

Biodiagnostics with multiplex dyes

A new bioanalytical concept that uses the characteristic fluorescence lifetimes of new multiplex dyes as an identification parameter is presented. Several dyes can be distinguished at the same wavelength by recognition of the fluorescence lifetimes. It is shown that the identification can be achieved in a capillary gel electrophoresis system in a few milliseconds with a misclassification rate of 10-4. As an example the simultaneous detection of two antibodies at one wavelength is presented.

Counting single molecules in living cells at high resolution by spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) and coincidence analysis

Recently, we have shown that single fluorescent dye molecules within the diffraction limited detection volume can be counted by coincidence analysis. In combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM), polarization modulation and high-resolution colocalization we suggested to use these techniques for the structural and dynamic investigation of functional protein assemblies and molecular machines in cells. Here we present the application of these techniques within fixed and living cells since quantification and observation of protein assembly in-vivo is of great interest for biological research. We show that appropriately chosen dyes, e.g. ATTO 620, can be discriminated from autofluorescent background within the cells by determination of their spectral emission and their fluorescence lifetimes measured by time correlated single photon counting (TCSPC) under pulsed laser excitation on a confocal microscope. Whereas a lot of autofluorescent signal can be found in the cytoplasm especially in living cells, the nucleus contains almost no fluorescent signal. This offers the opportunity to apply the above methods to protein assemblies, e.g. transcription units, within the cell nucleus. By investigation of fluorescently labeled poly-T40-oligonucleotides hybridized to poly-A-termini of mRNA or tethered within the cell nucleus we demonstrate the feasibility of coincidence analysis for counting single fluorescent molecules within fixed and living cells as a fundamental step for structural investigation below the diffraction limit of optical resolution.

In vitro phototoxicity of a new phthalocyanine-immunoconjugate for use in photodynamic therapy

Non specific localization of photosensitizers after application in vivo limits progress in PDT. Relatively selective distribution of photosensitizers in malignant tissues is crucial for a successful treatment. The target specificity of photosensitizers may be improved by linking photosensitizers with monoclonal antibodies. In this approach, a high specific monoclonal antibody, BM-2 which is directed against epitopes of the mucine glycoprotein TAG-12 was used. This antibody shows reactivity with 96% of all primary breast carcinomas. BM-2 was conjugated with a second generation phthalocyanine photosensitizer which is only weakly phototoxic to human T-47D tumor cells without conjugation in vitro. With respect to future clinical application, illumination times from 25 to 100 minutes and a powerful diode laser with an emission of 690 nm was chosen, which provides deeper tissue penetration in vivo. We observed phototoxicity towards T-47D human breast carcinoma cells at concentrations ranging from 0.25 to 6 micromol/L and light doses from 6 to 24 J/cm2. The immunoconjugates discriminated mucine-positive and mucine-negative tumor cells and showed high photocytotoxic selectivity towards mucine-positive T-47D cells in vitro. The conjugate showed no dark toxicity. In vivo experiments will follow.

KrF-excimer-laser-induced absorption and fluorescence bands in fused silica related to the manufacturing process

Transient and permanent UV absorption bands and fluorescence bands induced by 248 nm excimer laser radiation in fused silica are reported. It is shown that the permanent absorption measurements are not suitable to characterize the material with respect to transmission of high power laser pulses. In fused silica samples with high OH - content recovery of the 210 nm absorption band is observed after the end of irradiation. In samples with low OH - content no quick re covery is observed. 1 .