The investigation of the structure and dynamics of biomolecules and biomolecular assemblies in living cells is of current interest in molecular biology. Recent developments in single molecule fluorescence spectroscopy (SMFS) have opened ways for investigating the dynamics and stoichiometry of individual biomolecular complexes e.g., by application of single pair fluorescence resonance energy transfer (spFRET) with alternating laser excitation (ALEX), and by improved labels and labeling techniques. In the recent years, we have developed a set of techniques that allow the determination of the spatial distribution of single fluorescent molecules and their identification by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as the observation of the dynamics of individual molecules immobilized on surfaces. Based on SFLIM we currently focus on investigating the diffusion kinetics of biomolecules in living cells. By combining high-resolution confocal fluorescence microscopy of single molecules with fluorescence correlation spectroscopy (FCS) we seek to quantitate diffusion coefficients and concentrations of relevant fluorescently labeled biomolecules within living cells thereby visualizing the heterogeneous distribution of local mobilities in the sample. The simultaneously acquired fluorescence intensity and lifetime images can further be used for additional single point measurements for obtaining i.e., information about the stoichiometry of immobilized biomolecular complexes based on photon anti-bunching. In addition the simultaneous acquisition of multiple characteristic properties by SFLIM, like spectral emission bands and fluorescence lifetime, offers the opportunity to discriminate different fluorescent probes and autofluorescence.

We present a new method for precisely measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations. The method is based on a modified Fluorescence Correlation Spectroscopy (FCS)-setup which is robust against many artifacts that are inherent to standard FCS ^{1, 2}. The core idea of the new method is the introduction of an external ruler by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by ab initio calculations of resulting correlation curves and subsequent affine transformation of these curves to match the measured auto- and cross-correlation functions. The affine transformation coefficient along the time axis then directly yields the correct diffusion coefficient. This method is not relying on the rather inexact assumption of a 3D Gaussian shaped detection volume. We measured the diffusion coefficient of the red fluorescent dye Atto-655 (Atto-Tec GmbH) in water and compared the obtained value with results from Gradient Pulsed Field NMR (GPF-NMR).

We use time-resolved single molecule fluorescence detection (MSMD) to investigate the fluorescence dynamics of a mutant of the wild-type Green Fluorescent Protein (GFP) from Aequorea victoria, the folding enhanced GFP (FEGFP). The folding enhanced GFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. This variant shows increased thermal stability and the ability to retain its fluorescence when fused to poorly folding proteins. Here we apply one- (OPE) and two- (TPE) photon excitation on freely diffusing FEGFP molecules. Under OPE, single FEGFP molecules undergo fluorescence flickering in the time scale of μs and tens of μs due to triplet formation and ground-state protonation-deprotonation, respectively. OPE fluorescence lifetimes of single FEGFP molecules show evidence for the presence of different emitting species, the I and B forms of FEGFP chromophore. TPE single FEGFP molecules flicker in fluorescence in the time scale of μs due to singlet-triplet transitions of the chromophore. Two-photon excitation of single FEGFP molecules results in the creation of a photoconverted species with a fluorescence lifetime of 2.5 ns, a species which is bright enough to be detected at the single molecule level. Our results indicate FEGFP is a promising fusion reporter for intracellular applications when using OPE and TPE microscopy with single molecule sensitivity.

Single molecule measurements are generally made in conditions that depart from physiological conditions, such as with molecules excised from cells or even immobilized on surfaces. Such departures can easily cause measurements on biomolecules to be inexact. A tracking instrument to follow a single molecule's path in three dimensions inside a living cell would be a major step towards enabling single-molecule observations in physiological conditions. We describe an instrument that will extend the state of the art in single-molecule tracking technology, allowing extended observations of single particles as they diffuse and are transported. Computations show that our approach should be capable of tracking a protein-sized object diffusing at intracellular speeds for average times of over two seconds - long enough to track a typical fluorescent molecule from capture to photobleaching.

Methods for increasing the luminescence intensity of lanthanide macrocycles, Quantum Dyes^(R), by the Fluorescence Resonance Energy Transfer Enhanced Luminescence (FRETEL) effect in the solid state have been developed. A homogeneous solution containing the europium or terbium Quantum Dye and an excess of selected energy transfer species is evaporated to dryness, resulting in a thin film that surrounds and embeds the Quantum Dye or its conjugates. Under these conditions, in the presence of the gadolinium-thenoyltrifluoroacetonate complex as the energy transfer species, the luminescence of the europium Quantum Dye increased approximately 6-fold upon drying. However, the presence of a nonemitting lanthanide such as gadolinium is not always required for this effect. In studies employing the 2,6-pyridinedicarboxylate ion as the energy transfer species, where both the terbium and the europium Quantum Dyes could be simultaneously excited at 280 nm, the presence of gadolinium actually decreased the luminescence compared to that obtained with the 2,6-pyridinedicarboxylate alone. The simplest explanation for the FRETEL effect is that fluorescence resonance energy transfer occurs between the photo-trapping energy transfer species, either unbound or complexed with the nonluminescent gadolinium ion. The energy being finally transferred to the luminescent lanthanide ion complexes with consequent increase in emission intensity. This new method for the enhancement of luminescence intensity in the solid state has the significant advantage of eliminating the need for the previously required aqueous emulsion, which was difficult to make and transport.

Transition metal complexes such as ruthenium complexes, having metal-to-ligand charge transfer (MLCT) states, are extensively used in solar energy conversion and electron transfer in biological systems and at interfaces. The dynamics of metal-to-ligand charge transfer and subsequent intermolecular, intramolecular, and interfacial electron transfer processes can be highly complex and inhomogeneous, especially when molecules are involved in interactions and perturbations from heterogeneous local environments and gated by conformation fluctuations. We have employed single-molecule spectroscopy, a powerful approach for studying inhomogeneous systems, to study the electron transfer dynamics of ruthenium complexes. We have applied a range of statistical analysis methods to reveal nonclassical photon emission behaviors of single ruthenium complexes, e.g., photon antibunching and photophysical ground-state recovering dynamics on a microsecond time-scale. The use of photon antibunching to measure phosphorescence lifetimes and single-molecule electron transfer dynamics at room temperature is demonstrated, which is a novel way of probing ground state regeneration in back electron transfer processes.

Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Fluorescence imaging and spectroscopy yield valuable and quantitative information about the optical properties and the spatial distribution of single molecules. Force spectroscopy by atomic force microscopy (AFM) or optical tweezers allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules. We present a combined AFM and total internal reflection fluorescence (TIRF) microscopy setup that enables ultrasensitive laser induced fluorescence detection of individual fluorophores, control of the AFM probe position in x, y and z-direction with nanometer precision, and simultaneous investigation of optical and mechanical properties at the single molecule level. Here, we present the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In future applications, fluorescence resonant energy transfer between single donor and acceptor molecules will be investigated.

We developed a photothermal method based on scattering around a nano-absorber that allows for the unprecedented detection of individual nano-objects such as gold nanoparticles with diameter down to 1.4 nm as well as CdSe nanocrystals. This method relies on the absorptive properties of the nano-object and does not suffer from the drawbacks of luminescence-based methods. We present here two different applications of this versatile detection method. First, we performed absorption spectroscopy of individual gold nanoparticles as small as 5nm and CdSe nanocrystals in the multiexcitonic regime. Second, we show the applicability of our method for new types of gold nanoparticles based DNA microarrays. In addition to the intrinsic signal stability due to the use of gold labelling, our technique does not require silver staining enhancement and permits to push the signal dynamics of such microarrays from the single nanoparticle detection to almost the full surface coverage.

The study of DNA-protein interactions is gaining increased attention due to their importance in cellular processes. Only a well-functioning interaction guaranties that such a process can take place without errors. So far, only a small percentage of these interactions have been unraveled, partially due to their complexity but also due to the fact that there are only a few techniques that permit the study of these interactions. In this report we describe the development of a research tool based on tethered bead motion and Resonance Light Scattering (RLS) from gold beads. This method permits the study of DNA-protein interactions and the screening of proteins binding to a specific DNA sequence.

We present the first observation, to our knowledge, of lasing from a levitated, dye droplet. The levitated droplets are created by computer controlled pico-liter dispensing into one of the nodes of a standing ultrasonic wave (100 kHz), where the droplet is trapped. The free hanging droplet forms a high quality optical resonator, which shape can be externally controlled by the ultrasonic field, yielding wavelength tunability and directional control of the emission. Our 700 nL lasing droplets consist of Rhodamine 6G dissolved in ethylene glycol, at a concentration of 0.02 M. In our experiments the droplets are optically pumped at 532 nm light from a pulsed, frequency doubled Nd:YAG laser, and the dye laser emission is analyzed by a fixed grating spectrometer. With this setup we have achieved reproducible lasing spectra in the wavelength range 610 nm - 650 nm. The lasing spectra can controllably be modulated by shaping the droplet. Lasing micro-droplets have been demonstrated earlier, where the droplets in free fall passed the pumping laser beam. The levitated droplet technique has successfully been applied for a variety of bio-analytical applications at single cell level. In combination with the lasing droplets, the capability of this high precision setup can further be applied to create a highly sensitive intra cavity absorbance detection system.

A new concept of fluorescence microscopy is presented allowing the breaking of the diffraction limit of optical microscopy by a factor of ca. five. It relies on measuring the temporal evolution of fluorescence after sudden switch-on of the light excitation. The observed temporal dynamics of the fluorescence signal can be converted into information about the spatial distribution of fluorophores within the exciting laser focus. The proposed scheme is technically simple and versatile, and allows resolution enhancement in all three dimensions.

F_oF₁-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F₁ part to the proton translocation through the F_o part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F_o motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ε-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ε-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ε resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ε, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F_o part.

The dynamical behavior of individual enzymes is studied by confocal microscopy. This technique makes it possible to gain insights into the detailed spectrum of molecular conformational changes and activities. We report the direct observation of a single CalB lipase-catalyzed reaction for extended periods of time (hours). A model in which a broad spectrum of conformations is involved is has to be invoked in order to explain the stretched exponential behavior of the off-times histograms.

There is a risk of contamination of surgical instruments by nfectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect ~100 zeptomoles/mm² with an area scan speed of ~20 cm²/s and for using the system to detect other agents of biomedical interest. A theoretical analysis and experimental measurements will be discussed.

We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.

There is a need for low cost, miniature, integrated optical systems for bioassay monitoring to meet the growing in vitro and point-of-care diagnostics markets. To this end, we are investigating the use of silicon photomultipliers (SiPM) as device upon which to base our technology development. SiPMs have been used successfully in many high-energy physics applications, but their application as a fully integrated biological detection platform has not been shown. In this paper we will present a new detection platform for the measurement of fluorescent biomolecules at much lower concentrations than commercially available systems. Our results show approaches that demonstrate the use of SiPM for the detection of fluorescent proteins and fluorescent-labelled DNA sequences. The SiPM and sample platforms are integrated so that the minimum distance separating the detector from the sample is realised. In addition, direct immobilisation of the DNA sequences onto the SiPM surface is achieved. This combined approach shows improved sensitivities for both the fluorescent proteins and fluorescent-labelled DNA. We are presenting results that show the use of SiPM as a successful technology for the measurement of fluorescent biomolecules at improved lower concentrations.

We have recently developed a wide-field photon-counting detector having high-temporal and high-spatial resolutions and capable of high-throughput (the H33D detector). Its design is based on a 25 mm diameter multi-alkali photocathode producing one photo electron per detected photon, which are then multiplied up to 10⁷ times by a 3-microchannel plate stack. The resulting electron cloud is proximity focused on a cross delay line anode, which allows determining the incident photon position with high accuracy. The imaging and fluorescence lifetime measurement performances of the H33D detector installed on a standard epifluorescence microscope will be presented. We compare them to those of standard single-molecule detectors such as single-photon avalanche photodiode (SPAD) or electron-multiplying camera using model samples (fluorescent beads, quantum dots and live cells). Finally, we discuss the design and applications of future generation of H33D detectors for single-molecule imaging and high-throughput study of biomolecular interactions.

New techniques for the early detection of cancer are fast emerging. This is essential for more effective diagnosis and control of the disease. We have used a High Performance Liquid Chromatography-Laser Induced Fluorescence (HPLCLIF) technique to record chromatograms of proteins in serum and ovarian tissue samples. The recorded chromatograms of normal, benign and malignant samples were analyzed using statistical (Principal Component Analysis) methods. It is shown that chromatograms of the samples can be classified into sets, and a model based on such a classification can be used to analyze protein profiles of test samples of serum and ovarian tissue for the detection of malignancies.

In this work we report Single Molecule (SM) microscopy studies of binding events between individual surface immobilized antibodies and fluorescent antigens using Total Internal Reflection (TIR) microscopy. Specific binding events of single biotin-conjugated 1quantum dots to anti-biotin antibodies, immobilized on an amine-coated cover glass using a heterobifunctional photo-reactive cross-linker were observed. The methodology of calculating the binding affinity (dissociation constant) from the time series of 2-D images is described.

We have begun developing an innovative ultra-fast single-photon counting imager which comprises a mega-pixel CMOS array and a newly-designed Image Intensifier. It is expected to have single photon sensitivity with 100 psec time resolution, operational at a total counting rate exceeding 1MHz. The readout is based on dead-time-free flash ADC, running at 1-2GS/s, followed by a FPGA for real-time parallel data processing. Such a device has not been realized before and is expected to revolutionize time-resolved fluorescence imaging and spectroscopy from a single-molecule to whole animal level. To evaluate the design principle, an Image Intensifier with a GaAsP photocathode (>40% quantum efficiency at 400-600 nm) followed by double MCP was evaluated together with an existing CMOS camera. In our future design, the image from CMOS Camera will be combined with the MCP output, followed by a set of FPGA and CPU for real time data processing. This stream line method will allow ultra fast single-photon counting with 100 psec time resolution and 20 μm position resolution (1M pixel imaging). In this paper, we present the design principle and preliminary results on its performance. Our future plan and the design goals are also described.

We have developed a practical method to control the number of attaching biomolecules to an AFM tip. Monolayer of OEG that has little interaction with some biomolecules was self-assembled on tips. Electric pulses were used to oxidize a patch of OEG monolayer on tip and generate COOH groups for further linking at the activated area. The surface groups were detected by chemical force titration. The total numbers of molecule bound on the tip was examined by the studies of biotin/streptavidin system.

For future fully integrated sensing applications, a CMOS sensor will be required. New CMOS photon counting sensors have recently become available and these devices provide high quantum efficiency, photon counting sensitivity, low power and new devices in arrays and with on-chip electronics. In biological applications, photon counting is focused on the detection of low intensity fluorescence signals from fluorophores conjugated to proteins or nucleic acid biomarkers from fluorescent proteins. We describe the development of a novel microtitre plate reader format, or bioassay platform that incorporates arrays of photon counting detectors for multiple parallel readout and data acquisition. Using Pyrex wafers, we have designed and fabricated custom-made reaction wells using Pyrex and deep ion trench etched silicon, which produce optically clear structures to facilitate fluorescence detection in biological samples volumes of 2 nL to 2 μL. For initial verification of the system, a new photon counting detector from SensL is used to determine the effectiveness of the wells as the bioassay platform. The compact unit consists of a fibre coupled silicon photon counting sensor, thermoelectric cooler, thermoelectric controller, active quenching circuit, power supplies, and an USB interface to the operating software. Included in the module is a counter with time binning capability. Sensitivity increases of more than two orders of magnitude in fluorescence detection are expected over commercially available instruments. This system demonstrates that a miniaturized, low cost solution is possible for fluorescence bioassay detection, which can be used to meet growing demands in the in vitro diagnostics and Point of Care markets.

Surface plasmon-coupled emission (SPCE) has been used to reduce the detection volume in fluorescence measurements. The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With the excitation through the glass prism at SPR angle (Kretschmann configuration), the detection volume is a composition (product) of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that using Kretchmann configuration in a microscope with high numerical aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL, which is necessary to observe a single cross-bridge in the muscle. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be also used to study the small conformational changes of macromolecules.

Optical microsphere resonators can function as highly sensitive bio/chemical sensors due to the large Q-factor, which leads to high light-matter interaction. The whispering gallery modes (WGM) arise at the surface of the microsphere, creating a highly enhanced optical field that interacts with matter on or near the microsphere surface. As a result, the spectral position of the WGM is extremely sensitive to refractive index changes near the surface, such as when bio/chemical molecules bind to the sphere. We show the potential feasibility of a microsphere ring resonator as a sensor for small molecules by demonstrating detection of sub-femtomole changes in SiO₂ molecules at the surface of the microsphere. In this experiment, the silica molecules act as an excellent model for small molecule analytes because of their 60 Dalton molecular weight, and because we know nearly the exact quantity of molecules at the surface, which enables a sensitivity characterization. We measure the spectral shifts in the WGMs when low concentrations of hydrofluoric acid (HF) are added to a solution that is being probed by the microsphere. As the HF molecules break apart the SiO₂ molecules at the sphere surface, the WGMs shift due to the sub-nano-scale decrease in the size of the microsphere. These calculations show that the sensitivity of this microsphere resonator is on the order of 500 attomoles. Our results will lead to the utilization of optical microspheres for detection of trace quantities of small molecules for such applications as drug discovery, environmental monitoring, and enzyme detection using peptide cleavage.

Optical methods and proteomics investigations are becoming promising approaches for early detection of many diseases, which remain clinically silent for long periods. We have used efficient High Performance Liquid Chromatography (HPLC) separation combined with highly sensitive laser induced fluorescence detection of proteins present in clinical samples for diagnostic applications in cervical cancer. The protein profile and the fluorescence of individual proteins were simultaneously recorded using our HPLC-LIF system. Protein profiles (Chromatogram) of serum from normal male and female volunteers with and without tobacco habits, and malignant serum samples were studied. Protein profiles were also recorded for lysates of exfoliated cells collected from Pap smear of normal and cancer patients. The protein profile patterns were subjected to Principal component Analysis. Discrimination of normal and malignant samples were achieved with very high sensitivity and specificity.

The detection and identification of single molecules represent one of the ultimate goals of analytical chemistry. We have designed, developed and tested a new family of photodetectors with Internal Discrete Amplification (IDA) mechanism. These photodetectors can operate in linear (analog) detection mode with gain-bandwidth product up to 5.10¹⁴ and one- or few-photon sensitivity, as well as in the photon counting mode with count rates up to 10⁸ cps. Their key performance characteristics exceed those of photomultiplier tube (PMT) and avalanche photodiode (APD) devices. The measured parameters of the detectors are: gain > 10⁵, excess noise factor as low as 1.02, maximum count rate > 10⁸ counts/s, and rise/fall time < 300 ps. The new family of the photo detectors may become an ideal solution for the problems of ultrasensitive and single-molecule detection by fluorescence spectroscopy and other optical methods.

The triplet state characteristics of Cy5 molecule structurally related to the trans-cis isomerization are studied by means of ensemble and single molecule measurements. Several new deactivations of the singlet state are identified. The delayed fluorescence and phosphorescence from Cy5 molecule were observed. Spectral evidences show that the dim state could originate from the cis-Cy5 with lower excitation rate. The power dependence analysis for the same individual Cy5 molecule indicates an efficient reverse intersystem crossing happened while the T₁-T_n absorption is in resonance with the excitation wavelength.