Karl H. Drexhage

Fluorescence quantum yield of oxazine and carbazine laser dyes

Abstract The structural features responsible for radiationless deactivation in Oxazine and Carbazine dyes are determined by measurement of the fluorescence quantum yield in various solvents and at different temperatures. A distinction can be made between temperature-independent radiationless processes involving H-vibrations and temperature-dependent processes which have their origin in a lack of rigidity of the molecular skeleton.

New fluorescent dyes in the red region for biodiagnostics.

The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

Structural relaxation of rhodamine dyes with different N-substitution patterns: A study of fluorescence decay times and quantum yields

Abstract The viscosity- and temperature-controlled dynamical behaviour of rhodamine dyes in the excited state has been investigated by stationary and time-resolved fluorescence measurements using synchrotron radiation. An efficient deactivation pathway is linked with rotation of the amino groups presumably towards a state with charge localization. It is shown how electronic and steric factors influence the relaxation rate.

TIME-RESOLVED DETECTION AND IDENTIFICATION OF SINGLE ANALYTE MOLECULES IN MICROCAPILLARIES BY TIME-CORRELATED SINGLE-PHOTON COUNTING (TCSPC)

A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the...

Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level

Abstract Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the μs- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.

New fluorescent markers for the red region

Two new classes of fluorescent dyes have been developed as labels for the red region of the spectrum: amide-bridged benzopyrylium dyes and carbopyronin dyes. The fluorescence quantum yield ranges from 20 to 90%, the decay time from 1 to 4 ns. The pH- and solvent-dependence of absorption and fluorescence are described in detail. Covalent attachment is possible via activated carboxyl groups.

Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser

Abstract We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).

Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers

We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1:05 0:33 ns (Cy5-dCTP), 2:07 0:59 ns (MR121-dUTP) and 3:88 1:71 ns (Bodipy-dUTP).

Diode laser based detection of single molecules in solutions

Abstract Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.

Fluorescence pattern recognition for ultrasensitive molecule identification: comparison of experimental data and theoretical approximations

Abstract A concept taken from information theory is applied to ultrasensitive fluorescence detection and identification of molecules. The theoretical misidentification probability is compared to the experimental error probability. It is found that 350 photons are sufficient to distinguish dye molecules with nearly identical absorption and emission properties but with different fluorescence lifetimes with an accuracy of 1 misidentification in 10 4 experiments. Thus we demonstrate the experimental usefulness of the Kullback-Leibler minimum discrimination information for the identification of molecules.