M. Sauer

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...

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-varying photon probability distribution of individual molecules at room temperature

Abstract The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time.

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.

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.

Probing conformational dynamics by photoinduced electron transfer

We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. Single-pair fluorescence resonance energy transfer (FRET) experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angströms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. We present data about structural changes of single biomolecules like DNA hairpins and peptides by using quenching electron transfer reactions between guanosine or tryptophan residues in close proximity to fluorescent dyes. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

New Techniques for DNA Sequencing Based on Diode Laser Excitation and Time-Resolved Fluorescence Detection

In the foreseeable future the complete sequencing of all 3 x 109 base pairs of the human genome will be finished using Sangers enzymatic chain termination method [1] in combination with various automated DNA sequencing machines [2 -10]. However, to understand the function of each gene and the corresponding health implications, genetic variations in different cell types, individuals, and organisms have to be investigated. Hence, alternative methods have to be developed that are even faster, more efficient, more accurate, and more costeffective. Among these new methods are capillary array electrophoresis [11-17], mass spectrometry [18], sequencing by hybridization [19-23], and singlemolecule sequencing [24-31]. Probably, the human genome will be sequenced before one of these techniques is widely used. However, they will provide a powerful tool for biological scientists in the twenty-first century. In the following sections of this chapter we restrict our discussion on new DNA sequencing methods with fluorescence-based detection and identification schemes.

Improving single-molecule FRET measurements by confining molecules in nanopipettes

In recent years Fluorescence Resonance Energy Transfer (FRET) has been widely used to determine distances, observe distance dynamics, and monitor molecular binding at the single-molecule level. A basic constraint of single-molecule FRET studies is the limited distance resolution owing to low photon statistics. We demonstrate that by confining molecules in nanopipettes (50-100 nm diameter) smFRET can be measured with improved photon statistics reducing the width of FRET proximity ratio distributions (PRD). This increase in distance resolution makes it possible to reveal subpopulations and dynamics in biomolecular complexes. Our data indicate that the width of PRD is not only determined by photon statistics (shot noise) and distance distributions between the chromophores but that photoinduced dark states of the acceptor also contribute to the PRD width. Furthermore, acceptor dark states such as triplet states influence the accuracy of determined mean FRET values. In this context, we present a strategy for the correction of the shift of the mean PR that is related to triplet induced blinking of the acceptor using reference FCS measurements.

Single-molecule spectroscopy to probe competitive fluorescence resonance energy transfer pathways in bichromophoric synthetic systems

Using single molecule fluorescence spectroscopy we have investigated fluorescence resonance energy transfer (FRET) occurring between two peryleneimide (PI) chromophores in a series of synthetic systems: PI end-capped fluorene trimers, hexamers and polymers for which the interchromophoric distance vary from 3.4 to 5.9 and 42 nm, respectively. By monitoring in parallel the fluorescence intensity and the number of independent emitting chromophores from each molecule, we could discriminate between competitive Foerster-type energy transfer processes such as energy hopping, singlet-singlet annihilation and singlet-triplet annihilation for the PI end-capped fluorine compounds. Due to different energy transfer efficiencies, variations in the interchromophoric distance enable switching between these processes. The single molecule fluorescence data reported here suggest that similar energy transfer pathways have to be considered in the analysis of single molecule trajectories of donor/acceptor pairs, as well as in the case of more complex systems like natural multichromophoric systems, such as light harvesting antennas or oligomeric fluorescent proteins.