The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.

Single molecule fluorescence detection of Atto590 in poly(vinyl alcohol) was achieved by using a wide-field epifluorescence microscope with CCD-camera detection. Image sequences are obtained from which the time traces of the detected molecules are built. We find a distinctive difference between the time evolution of the fluorescence originating from the molecules detected in the first image of the sequence compared to the time evolution of the fluorescence of the molecules detected in each image of the sequence. Atto590 shows very long blinking times and photobleaching and photoblinking that are both quadratically dependent on the irradiation power density. Our approach allows kinetic separation of photobleaching from blinking. The possibility of choosing different ensembles of molecules is demonstrated and taken advantage of for this aim. Initially dark molecules or low emitting ones that might be overlooked are important to describe the complete ensemble behavior.

A simple imaging method for direct determination of single-molecule orientations is presented that uses a wide-field epifluorescence microscope and a sensitive CCD camera. Imaging is performed with slight defocusing of the optics, allowing for direct determination of single-molecule orientation based on the characteristic intensity distribution of the defocused images. Exact wave-optical calculations of these defocused images are presented and are in good agreement with the measurements. These calculations represent what is to the authors' knowledge the first complete wave-optical modeling of defocused imaging of dipole emitters at an interface; the peculiarities of dipole emission at an interface and the vector effects of that emission and of imaging with a high-numerical-aperture objective are taken into account. (C) 2003 Optical Society of America.

Fluorescence correlation spectroscopy (FCS) has developed into a widely used and very successful spectroscopic technique for detecting and quantifying minute concentrations of fluorescing dyes in solution, as well as for obtaining information about molecular parameters such as diffusion coefficients, photophysical transition rates, or chemical reaction kinetics. The standard evaluation method of FCS data is based on the simplified assumption that the convolution of the excitation intensity distribution and the light collection efficiency function is a three-dimensional Gaussian distribution. Although that assumption leads to satisfactory fits of experimental data, the adjustable parameters of this standard model have a rather unphysical meaning. In the present paper, an ab initio approach to modeling FCS curves is developed that is based on exact wave-optical calculations of fluorescence excitation and detection. Comparison with the standard model is made, and the ab initio calculated model curves are used for fitting experimental data and deriving absolute values of diffusion coefficient and concentration.

We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.

Recently Török and colleagues published in a series of papers a general wave-optical approach to calculating the light-collection efficiency function (CEF) for confocal imaging of dipole emitters [J. Mod. Opt. 45, 1681 (1998); J. Microec. 194, 127 (1999); Opt. Lett. 25, 1463 (2000)]. In their theory they did not address the possibility that the lifetimes of fluorescing molecules can change significantly near interfaces, which has a direct effect on the CEF. The research of Török and colleagues is extended here to include this effect, which may become important for imaging near surfaces.

This review presents an overview of the fluorescence detection and spectroscopy of single molecules (SMS) in liquids and on surfaces under ambient conditions. The various techniques of SMS, such as confocal epifluorescence detection and wide-field imaging are presented and discussed, together with the different methods of data analysis such as fluorescence correlation spectroscopy and burst-by-burst analysis. Selected applications of the various techniques in physics, chemistry, and biology are described.

The design of fluorescent probes (and labels) is as challenging as it ever was. Such probes enable studies on the molecular dimensions and dynamics of even complex (bio)matter, but also bioanalytical and screening assays whose sensitivity can reach the single molecule level. The design of advanced labels for bioassays is paralleled by developments in (laser) fluorescence spectroscopy, opto-electronics and data processing. Light-emitting diodes (LEDs) and diode lasers (DLs) are particularly attractive light sources and we therefore have focused our research (a) on labels that are LED- or DL-compatible, and (b) on applications of such labels to various analytical formats.In this article, we give an overview of our recent activities in the following areas: (1) a general logic for designing fluorescent probes and labels; (2) new diode laser-excitable probes for non-cocalent protein detection; (3) diode laser-compatible amino-reactive covalent labels; (4) diode laser-assisted fluorescent single molecule detection of dyes and labeled proteins; (5) new labels for flow cytometric determination of HSA; (6) new DNA labels; (7) fluorescence resonance energy transfer gene assays; (8) reactive ruthenium ligand complexes as markers for bioassays; (9) diode laser-excitable fluorescent polymer beads; (10) polyaniline-coated nanobeads as fluorescent pH probes; (11) phosphorescent poly(acrylonitrile) nanospheres as markers for optical assays; (12) competititve binding of streptavidin to biotinylated nanobeads as studied by resonance energy transfer; (13) nanobeads as reference dyes in luminescent lifetime imaging using DLR; (14) phosphorescent nanospheres for use in advanced time-resolved multiplexed bioassays; (15) beads dyed with a europium-based label and excitable with the 405-nm diode laser; and (16) a europium(III)-based probe for use in oxidase-associated reactions.

Fluorescence lifetime measurement of organic fluorophores is a powerful tool for distinguishing molecules of interest from background or other species. This is of interest in sensitive analysis and Single Molecule Detection (SMD). A demand in many applications is to provide 2-D imaging together with lifetime information. The method of choice is then Time-Correlated Single Photon Counting (TCSPC). We have devloped a compact system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner holding the immobilized sample. The system allows count rates up to 3 MHz and a resolution down to 30 ps. An overall Instrument Response Function down to 300ps is achieved with inexpensive detectors and diode lasers. The board is designed for the PCI bus, permitting high throughput without loss of counts. It is reconfigurable to operate in different modes. The Time-Tagged Time-Resolved (TTTR) mode permits the recording of all photon events with a real-time tag allowing data analysis with unlimited flexibility. We use the Time-Tag clock for an external piezo scanner that moves the sample. As the clock source is common for scanning and tagging, the individual photons can be matched to pixels. Demonstrating the capablities of the system we studied single molecule solutions. Lifetime imaging can be performed at high resolution with as few as 100 photons per pixel.

A new method of performing fluorescence correlation spectroscopy (FCS) measurements for mixtures of several fluorescent molecular species is introduced. It uses time-resolved fluorescence detection for separating the different FCS-contributions from the different species. This allows simultaneous and independent monitoring of the diffusion of several molecular species in one sample, or performing multi-label cross-correlation measurements. In this way, the proposed method is equivalent to dual- or multi-color FCS. However, it is simpler to implement experimentally, because it requires only single wavelength excitation and detection. This Letter outlines the theoretical basis and presents experimental results of the method.

In this research, a near-IR fluorescent labeled nucleic acid conjugate for the nucleic acid detection was synthesized, and characterized preliminarily for the detection of the nucleic acid. Th conjugate combines the molecular recognition properties of the oligonucleotides with the near-IR fluorescence label PR646. Both single- and dual- labeled conjugates were studied for their hybridization with the complementary nucleic acid. The dual labeled conjugate has indicated that the self-quenching effect exists in ssDNA form while the fluorescence increases greatly after hybridization with the complementary nucleic acids. The time-resolved fluorescence was also studied.

We describe a series of new long-wave absorbing and fluorescing cyanine dyes and labels (based on a general logic for the design of such dyes), their spectra, covalent and noncovalent linkage to proteins, their use in single molecule detection (SMD) and as donors and acceptors, respectively, in fluorescence resonance energy transfer studies. The new labels represent water-soluble and reactive fluorophores whose quantum yields increase substantially if noncovalently or covalently bound to proteins. Due to their strong absorptions between 550 and 700 nm they are excitable by light-emitting diodes or diode lasers. Their high absorbances (epsilon around 100000) and adequate fluorescence quantum yields (phi up to 0.68 if bound to proteins) along with their availability as reactive NHS esters make them viable labels for proteins and oligomers, e.g. in context with SMD or fluorescence energy transfer immunoassay which is demonstrated for the system HSA/anti-HSA.

A confocal laser-scanning microscope for ultrasensitive fluorescence lifetime imaging on surfaces is presented. The system employs a compact electronics for time-correlated single-photon counting (TCSPC), allowing for measuring fluorescence lifetime with 40 ps time resolution, and for continuously recording photon arrival times with 100 ns time resolution. Additionally developed driver electronics serve for synchronization of scanning and data acquisition, which is significant for achieving high spatial image resolution. The capabilities of the measurement system are demonstrated on imaging single molecules immobilized on glass substrates. Finally, it is shown how the TCSPC capabilities of the system can be used not only for lifetime imaging but also for multichannel measurements.

In recent years, laser induced fluorescence detection and spectroscopy of single molecules (SMD) has seen a tremendous development. Besides fundamental research on individual molecular systems, practical applications of this sensitive detection technique for analytical and diagnostic purposes are becoming more and more important. For a wider applicability of the SMD technique it is desirable to detect not only the presence or absence of a molecule within a given detection volume, but also to be able to quantify this fluorescence absolutely. Modified now cytometry systems for SMD that are ensuring such a quantifiable fluorescence detection of single molecules are already successfully applied for e.g. DNA fragment sizing. In our talk, we present a modified confocal detection set-up for performing SMD on surfaces that aims at a quantified detection of single molecule fluorescence. First experimental results presented and emerging problems are discussed.

We present a comparison between two basically different optical detection systems: a confocal epifluorescence microscope, and a new evanescent wave detection system employing a parabolic optical element. In a microscope set-up, fluorescence light is collected within a cone around the optical axis, whereas in the evanescent light detector, fluorescence light is collected mainly at angles larger than the so-called critical angle of total internal reflection. Based on a thorough theoretical modeling of both experimental set-ups, comparison between the two detection systems is made Particularly, the optical detection efficiency is compared. 5