This PDF file contains the front matter associated with SPIE Proceedings Volume 7185, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing

Enzymes engage key roles in a wide variety of important physical and medical processes, which thus can be altered by manipulating the behavior of enzymes in charge. The capability for manipulation requires an exact understanding of enzymatic operation modes though, which can be increased by employing fluorescence spectroscopy techniques. To date several fluorescence-based assays using labeled substrates have been developed to examine different subclasses of hydrolases. We developed a method that circumvents the unspecific probe enzyme interactions and affinity problems occurring in common probes as those based on fluorescence resonance energy transfer (FRET) by taking advantage of the comparably strong electron donating properties of the naturally occurring nucleic acid guanosine (G). Combined with an appropriate fluorophore this compound shows efficient photoinduced electron transfer (PET) quenching reactions only upon contact formation. Thus, initially quenched enzyme substrates, e.g. specific nucleic acid sequences, can be designed that cause a distinct increase in fluorescence signal upon specific hydrolysis. Here we demonstrate the general validity of PET probes for the observation of various nucleases at the ensemble and single molecule level. The rapid response time of the probes enables real-time monitoring of enzyme activities and provides quantitative data which are compared to those of commonly available and recently published, more complex probes. Additionally the applicability of this method is demonstrated for peptidases via fluorophore tryptophan (Trp) interaction.

We investigate experimentally the modifications of the fluorescence properties of the bichromophoric fluorescent resonance energy transfer (FRET) system DsRed imposed by optical confinement. The confinement-condition is realized by a novel λ/2-microresonator that modifies the local photonic mode density in the vicinity of the proteins while maintaining a physiological environment for the embedded biological molecules. The experimental ratio of the fluorescence intensities and lifetimes, respectively, of donor and acceptor chromophores varies by up to a one order of magnitude as we vary the mirror spacing of the microresonator with nanometer-precision. Since these ratios determine the FRET efficiency, we modify the yield of the excited state energy transfer in rigidly coupled FRET pairs without chemically or physically perturbating the chromophoric subunits. We show that the microresonator-controlled inhibition of the acceptor fluorescence results in a loss of transfer efficiency of excited state energy from donor to acceptor, an effect that enables the spectral isolation and efficient observation of donor chromophores both in DsRed ensembles and on the single protein level. This constitutes an important application of microcavity-enhanced single molecule spectroscopy of biological systems and shows the potential of optical confinement for applications in nano-biophotonics.

F_oF₁-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound F_o part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ε subunits of the F₁ sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F₁ motor, another one to the F_o motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of F_o and F₁.

Electron and energy transfer in proteins are key processes in bioenergetics. Their understanding on a molecular level can serve as an important guideline for the design of nanoscale assemblies. Energy transfer between pigment molecules requires a match between their transition energies for energy emission and absorption. The tuning of these pigment energies in proteins is achieved by pigment-protein interactions. In general, these interactions are regarded as static properties determined by the three-dimensional structure of pigment-protein complexes. Employing single-molecule fluorescence spectroscopy we demonstrate that protein dynamics, even at cryogenic temperatures, significantly influences the transition energy of pigments and, as a consequence, modulates energy transfer pathways. This variability of excitation energy transfer pathways introduced by protein dynamics might be important for the extreme robustness of photosystems.

We performed quantitative tests in order to compare the practical limits of FCS and FLCS. Unlike conventional FCS, FLCS yields precise and correct concentration values from as low as picomolar to micromolar concentrations. We discuss some of the inherent technical limitations of FCS and demonstrate that they are easily overcome by FLCS employing the simplest confocal detection scheme.

We have previously shown that formation of triplet states and other photo-induced states can be controlled by modulating the excitation with pulse widths and periods in the range of the transition times of the involved states. However, modulating the excitation in fluorescence correlation spectroscopy (FCS) measurements normally destroys correlation information and induces ringing in the correlation curve. We have introduced and experimentally verified a method to retrieve the full correlation curves from FCS measurements with modulated excitation and arbitrarily low fraction of active excitation. Modulated excitation applied to FCS experiments was shown to suppress the triplet build-up more efficiently than reducing excitation power with continuous wave excitation. The usefulness of the method was demonstrated by measurements done on fluorescein at different pH, where suppression of the triplet significantly facilitates the analysis of the protonation kinetics. Using a fluorophore where the protonation-coupled fluorescence intensity fluctuations are due to spectral shifts, introduction of two-color alternating excitation and spectral crosscorrelation can turn the protonation component of the correlation curve into an anti-correlation and further facilitate the distinction of this component from those of other processes.

Fluorescence detection is a central component in biological research. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. A single-stranded oligonucleotide was chemically bound to a single 50 nm diameter silver particle and a Cy5-labeled complementary single-stranded oligonucleotide was hybridized with the particle-bound oligonucleotide. The bound Cy5 molecules on the silver particles were spatially separated from the silver surface by the hybridized DNA duplex chains, which were about 8 nm in length, to reduce the competitive quenching. We use fluorescence lifetime correlation spectroscopy (FLCS) with picosecond time-resolved detection to separate the fluorescence correlation spectroscopy (FCS) contributions from fluorophores and metal-conjugated fluorophores. The single Cy5-labeled 50 nm silver particles displayed a factor of 15-fold increase in emission signal and 5-fold decrease in emission lifetimes in solution relative to the Cy5-DNA in the absence of metal. Lifetime measurements support the near-field interaction mechanism between the fluorophore and silver nanoparticle. In this study, FLCS is being applied to a system where the brightness and the fluorescent lifetime of the emitting species are significantly different. Our measurements suggest that FLCS is a powerful method for investigating the metal-fluorophore interaction at the single molecule level and to separate two different species from a mixture solution emitting at the same wavelength. Additionally, the highly bright Cy5-DNA-Ag molecules offer to be excellent probes in high background biological samples.

We present the results on two-photon total-internal-reflection fluorescence correlation spectroscopy. The combination of liquid crystal spatial light modulator, providing radial polarization, with ultrafast laser (picosecond Nd:GdVO₄ laser) allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

High-resolution fluorescence imaging has a vast impact on our understanding of intracellular organization. The key elements for high-resolution microscopy are reversibly photo-switchable fluorophores that can be cycled between a fluorescent and a non-fluorescent (dark) state and can be localized with nanometer accuracy. For example, it has been demonstrated that conventional cyanine dyes (Cy5, Alexa647) can serve as efficient photoswitchable fluorescent probes. We extended this principle for carbocyanines without the need of an activator fluorophore nearby, and named our approach direct stochastic optical reconstruction microscopy (dSTORM). Recently, we introduced a general approach for superresolution microscopy that uses commercial fluorescent probes as molecular photoswitches by generating long lived dark states such as triplet states or radical states. Importantly, this concept can be extended to a variety of conventional fluorophores, such as ATTO520, ATTO565, or ATTO655. The generation of non-fluorescent dark states as the underlying principle of superresolution microscopy is generalized under the term photoswitching microscopy, and unlocks a broad spectrum of organic fluorophores for multicolor application. Hereby, this method supplies subdiffraction-resolution of subcellular compartments and can serve as a tool for molecular quantification.

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10^-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

Novel methods of visible light microscopy have overcome the limits of resolution hitherto thought to be insurmountable. The localization microscopy technique presented here based on the principles of Spectral Precision Distance Microscopy (SPDM) with conventional fluorophores under special physical conditions allows to measure the spatial distribution of single fluorescence labeled molecules in entire cells with macromolecular precision which is comparable to a macromolecular effective optical resolution. Based on detection of single molecules, in a novel combination of SPDM and Spatially Modulated Illumination (SMI) microscopy, a lateral (2D) effective optical resolution of cellular nanostructures around 10 - 20 nm (about 1/50^th of the exciting wavelength) and a three dimensional (3D) effective optical resolution in the range of 40 - 50 nm are achieved.

Photon coincidence analysis is nowadays a widely used technique to study fluorescence intensity fluctuations, taking place on a timescale from seconds down to picoseconds. Photon bursts in the microsecond regime are e.g. used to study diffusion properties via Fluorescence Correlation Spectroscopy (FCS). Photon bunching in the microsecond regime allows to study fast conformational changes as well as internal photophysics like singulett-triplet transitions. Interphoton delay times in the ns regime carry information about the fluorescence lifetime and can also be used to characterise molecular rotation. Down in the picosecond regime, photon antibunching is used to quantify a small number of emitters and especially to proof the existence of a single emitting dye molecule. All of these methods can be carried out with the single molecule sensitive confocal fluorescence microscope MicroTime 200 and are based on time-correlated single photon counting (TCSPC). We developed a generalized approach to store the individual photon arrival time information with ps accuracy on a timescale up to hours which allows to study all mentioned phenomena in a single measurement (Full Correlation Analysis). Using the new HydraHarp 400 TCSPC unit we can now acquire photon information in 4 completely independent detection channels. This paper present the straightforward experimental concept as well as typical results and recent application examples.

Fluorescence correlation spectroscopy (FCS) has been extensively applied to study the kinetics and photophysics of molecules as well as interactions between molecules by extracting information from the fluctuation of signals. In particular, single molecule applications of FCS promise the greatest amounts of information. Ideally, one would like to carry out FCS in real-time; however, due to the time-consuming nature of the correlation process, performing the correlation in real-time is totally nontrivial. Generally an expensive hardware correlator or a TCSPC board is required for this purpose. Recently highly-efficient algorithms based on multi-tau method have been proposed to build up a software correlator. In this work, we set forth an innovative algorithm capable of realizing the real-time correlation, without turning to the multi-tau method. This algorithm takes advantage of the low count rate generally existing in the FCS experiments, directly using the time interval between each photon its adjacent photon to efficiently update the correlation function. Based on this efficiency, it is possible to build a low-cost software correlator with just an ordinary counter board. We practically demonstrate the feasibility by setting up this correlator to measure the diffusion motion of rhodamine 6G in water using FCS. The algorithm was validated by duplicating the signal from the photon detector and sending it to both the ordinary counter board with our software correlator and a commercial correlator simultaneously. The perfect coincidence of the correlation curves from these two correlators and the real-time display of the correlation function indicate the validity and practicability of our approach.

Fluorescence lifetime imaging (FLIM) is a powerful approach to studying the immediate environment of molecules. For example, it is used in biology to study changes in the chemical environment, or to study binding processes, aggregation, and conformational changes by measuring Förster resonance energy transfer (FRET) between donor and acceptor fluorophores. FLIM can be acquired by time-domain measurements (time-correlated single-photon counting) or frequency-domain measurements (with PMT modulation or digital frequency domain acquisition) in a confocal setup, or with wide-field systems (using time-gated cameras). In the best cases, the resulting data is analyzed in terms of multicomponent fluorescence lifetime decays with demanding requirements in terms of signal level (and therefore limited frame rate). Recently, the phasor approach has been proposed as a powerful alternative for fluorescence lifetime analysis of FLIM, ensemble, and single-molecule experiments. Here we discuss the advantages of combining phasor analysis with a new type of FLIM acquisition hardware presented previously, consisting of a high temporal and spatial resolution wide-field single-photon counting device (the H33D detector). Experimental data with live cells and quantum dots will be presented as an illustration of this new approach.

Creating a versatile set of highly stable fluorophores capable of high emission rate is crucial to studies of the individual function of biomolecules. There are continuing efforts to increase the sensitivity of fluorescence. These efforts include modifications in the spectral properties of the probes, increasing the detection efficiencies of the instruments, or the use of amplification methods. Our previous results show that plasmonic-controlled fluorescence provides a novel physical mechanism to enhance fluorescence intensity, reduce blinking and increase photostability. The further development of fluorophore-metal interactions for single molecule detection requires defined structures. For example, we investigate the effects of the defined silver nanospheres fabricated by wet chemistry methods coupling to nearby organic fluorophores. Additionally, we are developing nanoparticles incorporated into the Quantum Dot (QD) system. Coupling between the plasmon resonance effect and the quantum size effect of the QD or the organic fluorophore may develop new aspects of nano-composite material systems and also widen applications for noble imaging probes.

We study numerically the measurement of distances and distance fluctuations by photothermal correlation spectroscopy and coupled plasmon resonances. Gold nanoparticle dimers form a coupled longitudinal plasmon resonance in the absorption cross section, which strongly depends on distance. This new plasmon resonance can be advantageously used to heat the particles in a photothermal microscope. We calculate the distance dependence of the photothermal signal as a function of particle size and distance. The results demonstrate that the photothermal signal autocorrelation function stay single exponential even for large amplitude fluctuations and thus directly reveals the dynamics of the distance fluctuations without any corrections as required for fluorescence resonance energy transfer (FRET). Further, we show, that this type of distance detection provides distance measures beyond the accessible range of a few nanometers as in FRET.

A microfluidic single molecule fluorescence-based detection scheme is developed to identify target protein direct from cell lysate by using polyclonal antibody. Relative concentration of target protein in solution is determined by twodimensional (2D) photon burst analysis. Compared to conventional ensemble measurement assays, this microfluidic single molecule approach combines the advantages of higher sensitivity, fast processing time, small sample consumption and high resolution quantitative analysis.

It is generally accepted that molecular motors are utilizing the chemical energy of adenosine triphosphate (ATP) hydrolysis to convert it to the mechanical energy. A set of preliminary data demonstrates that the periodic electric field can induce transport as well, thus providing the energy to the molecular system.

The ability to follow and observe single molecules as they function in live cells represents a major milestone for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior studies of two dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008). In addition, the microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of following single molecule dynamics on live cells with three dimensional spatial resolution.

Interdependent structural properties such as molecular conformation, flexibility and charge redistribution control the intermolecular interactions of acetylcholine (ACh) with adjacent molecules. This paper reports the results of an investigation of the effect of the diffusion of ACh through a nano/microporous poly (N-vinylimidazole) (PVI) gel on its structural properties, namely on changes in its conformation. To investigate the conformational changes of ACh during spontaneous diffusion through the gel, the fluorescence lifetime of the label molecule - fluorescein - was monitored. To clarify the results, analogous experiments were conducted with nicotinic acid and dopamine. In contrast to the nicotinic acid and dopamine, ACh can play the role of a regulator in molecular transport.

To obtain specific biochemical information in optical scanning microscopy, labeling technique is routinely required. Instead of the complex and invasive sample preparation procedures, incorporating spectral acquisition, which commonly requires a broadband light source, provides another mechanism to enhance molecular contrast. But most current optical scanning system is lens-based and thus the spectral bandwidth is limited to several hundred nanometers due to anti-reflection coating and chromatic aberration. The spectral range of interest in biological research covers ultraviolet to infrared. For example, the absorption peak of water falls around 3 μm, while most proteins exhibit absorption in the UV-visible regime. For imaging purpose, the transmission window of skin and cerebral tissues fall around 1300 and 1800 nm, respectively. Therefore, to extend the spectral bandwidth of an optical scanning system from visible to mid-infrared, we propose a system composed of metallic coated mirrors. A common issue in such a mirror-based system is aberrations induced by oblique incidence. We propose to compensate astigmatism by exchanging the sagittal and tangential planes of the converging spherical mirrors in the scanning system. With the aid of an optical design software, we build a diffraction-limited broadband scanning system with wavefront flatness better than λ/4 at focal plane. Combined with a mirror-based objective this microscopic system will exhibit full spectral capability and will be useful in microscopic imaging and therapeutic applications.