C. Zander

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.

DETECTION AND CHARACTERIZATION OF SINGLE MOLECULES IN AQUEOUS SOLUTION

Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet stateS1 with a frequency-doubled titanium: sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100 ps were generated by convolution with the excitation pulse. The lifetime of theS1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79 ± 0.33 ns (RB) and 3.79 ± 0.38 ns (R6G) in aqueous solution.

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...

SINGLE-MOLECULE COUNTING AND IDENTIFICATION IN A MICROCAPILLARY

Abstract Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 μm at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser (λ=640 nm, average power 800 μW, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.

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.

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.

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.

Ultrasensitive Detection and Identification of Biomolecules with Diode Lasers - From Dyes to DNA

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 biological applications with absorption and emission beyond 600 nm. We applied the pattern recognition technique taken from information theory to ultrasensitive fluorescence detection and identification of dye molecules. Using pulsed diode lasers emitting at 630-640 nm in combination with new efficient rhodamine and oxazine dyes four-dye one-lane DNA sequencing in capillary gelelectrophoresis with time-resolved fluorescence detection is demonstrated.