Jutta Arden-Jacob

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.

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).

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.

New fluorescent labels for time-resolved detection of biomolecules.

New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of “multiplex dyes,” we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.

Characterization of new fluorescent labels for ultra-high resolution microscopy

Photo-induced switching of dyes into dark, long-lived states, such as a triplet state, has recently gained increasing interest, as a means to achieve ultra-high optical resolution. Additionally, these long lived states are often highly environment-sensitive and their photodynamics can thus offer additional independent fluorescence-based information. However, although providing a useful mechanism for photo-induced switching, the triplet state often appears as a precursor state for photobleaching, which potentially can limit its usefulness. In this work, a set of rhodamine and pyronin dyes, modified by substitution of heavy atoms and nitrogen within or close to the central xanthene unit of the dyes, were investigated with respect to their triplet state dynamics and photostabilities, under conditions relevant for ultra-high resolution microscopy. Out of the dyes investigated, in particular the rhodamine and pyronin dyes with a sulfur atom replacing the central oxygen atom in the xanthene unit were found to meet the requirements for ultra-high resolution microscopy, combining a prominent triplet state yield with reasonable photostability.

Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes

Abstract A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.

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.

Sensitive fluorescence detection using laser diodes and multiplex dyes

Abstract A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.