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We present an interferometric spectral-domain optical coherence tomography microscopy setup to detect structural changes using interference of light reflected from different interfaces of the sample. We induce a reproducible nanometer-scale size change in dye-doped 10-µm polystyrene microspheres by the release of Stokes shift energy of dye molecules inside the microspheres, excited by a modulated 532-nm laser. The resulting optical path length difference was measured with a sensitivity of 0.4 p m / H z limited by photodetection noise, and reveals elastic as well as inelastic responses, which opens up possibilities for measuring the response of cell-sized biological objects.

In this work, using a custom-built Spectral-domain optical coherence tomography microscopy setup we measured expansion caused by heat dissipation during fluorescence emission in dye-doped polystyrene microspheres with 70 pm sensitivity.

Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye‐labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold‐core rhodamine B‐labelled silica particles, 15 nm CdSe/CdS/ZnS core–shell–shell quantum dots (QDs) and 230 nm Y2O3:Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold‐core rhodamine B‐labelled silica NPs and QDs are quenched after a single exposure to 60 ke− nm–2 with an energy of 120 keV, while Y2O3:Eu3+ NPs are robust and still show luminescence after five doses of 60 ke− nm–2. In addition, the luminescence intensity of Y2O3:Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2O3:Eu3+ NPs are promising as robust fiducial marker in CLEM.

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (SignificanceWe have developed a correlative imaging pipeline to (i) correlate 3D-FM to volume-EM data with high precision, directly bridging the FM and EM resolutions (ii) achieve high-throughput volume-CLEM by targeted EM imaging of a single organelle sized region-of-interest, pre-identified by FM (iii) link live-cell fluorescence imaging of cultured mammalian cells to high-throughput volume-CLEM (iv) quantitatively study structure-function relations at subcellular scale (v) link rare (e.g. membrane contact sites) and transient (e.g. organelle interactions) cellular events to 3D ultrastructure.The targeted volume-CLEM pipeline provides a unique prospect for multi-modal correlative intracellular analysis combining dynamic interaction (live-cell imaging), functional state (live-cell imaging), molecular localization (FM), and 3D-ultrastructure (FIB.SEM) at nanometer scale.

In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy. CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided. Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm.

We present a universally applicable 3D‐printed external light trap for enhanced absorption in solar cells. The macroscopic external light trap is placed at the sun‐facing surface of the solar cell and retro‐reflects the light that would otherwise escape. The light trap consists of a reflective parabolic concentrator placed on top of a reflective cage. Upon placement of the light trap, an improvement of 15% of both the photocurrent and the power conversion efficiency in a thin‐film nanocrystalline silicon (nc‐Si:H) solar cell is measured. The trapped light traverses the solar cell several times within the reflective cage thereby increasing the total absorption in the cell. Consequently, the trap reduces optical losses and enhances the absorption over the entire spectrum. The components of the light trap are 3D printed and made of smoothened, silver‐coated thermoplastic. In contrast to conventional light trapping methods, external light trapping leaves the material quality and the electrical properties of the solar cell unaffected. To explain the theoretical operation of the external light trap, we introduce a model that predicts the absorption enhancement in the solar cell by the external light trap. The corresponding calculated path length enhancement shows good agreement with the empirically derived value from the opto‐electrical data of the solar cell. Moreover, we analyze the influence of the angle of incidence on the parasitic absorptance to obtain full understanding of the trap performance. © 2015 The Authors. Progress in Photovoltaics: Research and Applications published by John Wiley & Sons, Ltd.

The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.

Lanthanide ions are promising for the labeling of silica nanoparticles with a specific luminescent fingerprint due to their sharp line emission at characteristic wavelengths. With the increasing use of silica nanoparticles in consumer products, it is important to label silica nanoparticles in order to trace the biodistribution, both in the environment and living organisms. In this work, we synthesized LaPO4 nanocrystals (NCs) with sizes ranging from 4 to 8 nm doped with europium or cerium and terbium. After silica growth using an inverse micelle method, monodisperse silica spheres were obtained with a single LaPO4 NC in the center. We demonstrate that the size of the silica spheres can be tuned in the 25–55 nm range by addition of small volumes of methanol during the silica growth reaction. Both the LaPO4 core and silica nanocrystal showed sharp line emission characteristic for europium and terbium providing unique optical labels in silica nanoparticles of variable sizes. Electronic supplementary material The online version of this article (doi:10.1186/s11671-016-1465-y) contains supplementary material, which is available to authorized users.

Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (∼10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.

Lifetime and spectral imaging are complementary techniques that offer a non-invasive solution for monitoring metabolic processes, identifying biochemical compounds, and characterizing their interactions in biological tissues, among other tasks. Newly developed instruments that perform time-resolved spectral imaging can provide even more information and reach higher sensitivity than either modality alone. Here we report a multispectral lifetime imaging system based on a field-programmable gate array (FPGA), capable of operating at high photon count rates (12 MHz) per spectral detection channel, and with time resolution of 200 ps. We performed error analyses to investigate the effect of gate width and spectral-channel width on the accuracy of estimated lifetimes and spectral widths. Temporal and spectral phasors were used for analysis of recorded data, and we demonstrated blind un-mixing of the fluorescent components using information from both modalities. Fractional intensities, spectra, and decay curves of components were extracted without need for prior information. We further tested this approach with fluorescently doubly-labeled DNA, and demonstrated its suitability for accurately estimating FRET efficiency in the presence of either non-interacting or interacting donor molecules.

We demonstrate a universally applicable 3D-printed external light trap for solar cells. We placed a macroscopic external light trap made of smoothened, silver coated plastic at the sun-facing surface of different types of solar cells. The trap consists of a reflective parabolic concentrator on top of a reflective cage. The trap directs the light that is reflected upwards by the solar cell back towards the solar cell. Due to this retro-reflection the light traverses the solar cell several times resulting in a broadband absorption enhancement. We present a 15-27% efficiency enhancement for crystalline silicon, thin film nanocrystalline silicon and organic solar cells.

We report on the values of the two-photon excitation action cross-sections of commercially available enhanced cyan, green, yellow, and red fluorescent proteins. The two-photon absorption spectra are very similar in shape to those measured for one-photon absorption. However, they exhibit a significant blueshift, which is attributed to the participation of a vibrational mode in the two-photon absorption process. The two-photon spectra are compared to that of flavine mononucleotide, which constitutes the main source of autofluorescence in mammalian cells. The definition of a relative detection yield between the autofluorescent proteins and flavine allows us to quantify the applicability of autofluorescent proteins in two-photon single-molecule studies in living cells.

The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

Essentially all approaches to artificial molecular motors rely on diffusional stepping, i.e. the free energy input that powers the motor is used to rectify thermal motion. However, many models for biological motors, e.g. myosins or kinesins, include directed motion due to a “power stroke”. In this project we are employing a bottom-up approach to developing a relatively simple experimental model system, the “Inchworm”, with the intention to create the first artificial motor with a power stroke. Specifically, the Inchworm consists of a stretch of DNA confined inside a nanochannel, with ligand-gated repressor proteins immobilized on the nanochannel walls. By cyclically stretching and contracting the Inchworm DNA, via changes in buffer ionic strength, and simultaneously externally controlling the DNA repressor proteins’ binding and unbinding states, the motor is designed to achieve processive and unidirectional motion along the nanochannel. Here we report the status of this project: we have constructed the Inchworm DNA, expressed the ligand-gated repressor proteins and tested their specificity. In addition, we have developed a protocol for immobilization of the repressor proteins to the nanochannel walls and produced nanochannels (100-200 nm) in quartz, which can be interfaced with preexisting micro-fluidic systems.Acknowledgment: This work was supported by MONAD, HFSP, the Swedish Research Council and nmC@LU.

The potential of digital holography for complex manipulation of micron-sized particles with optical tweezers has been clearly demonstrated. By contrast, its use in quantitative experiments has been rather limited, partly due to fluctuations introduced by the spatial light modulator (SLM) that displays the kinoforms. This is an important issue when high temporal or spatial stability is a concern. We have investigated the performance of both an analog-addressed and a digitally-addressed SLM, measuring the phase fluctuations of the modulated beam and evaluating the resulting positional stability of a holographic trap. We show that, despite imparting a more unstable modulation to the wavefront, our digitally-addressed SLM generates optical traps in the sample plane stable enough for most applications. We further show that traps produced by the analog-addressed SLM exhibit a superior pointing stability, better than 1 nm, which is comparable to that of non-holographic tweezers. These results suggest a means to implement precision force measurement experiments with holographic optical tweezers (HOTs).

Molecular spiders are synthetic biomolecular walkers that use the asymmetry resulting from cleavage of their tracks to bias the direction of their stepping motion. Using Monte Carlo simulations that implement the Gillespie algorithm, we investigate the dependence of the biased motion of molecular spiders, along with binding time and processivity, on tunable experimental parameters, such as number of legs, span between the legs, and unbinding rate of a leg from a substrate site. We find that an increase in the number of legs increases the spiders' processivity and binding time but not their mean velocity. However, we can increase the mean velocity of spiders with simultaneous tuning of the span and the unbinding rate of a spider leg from a substrate site. To study the efficiency of molecular spiders, we introduce a time-dependent expression for the thermodynamic efficiency of a molecular motor, allowing us to account for the behavior of spider populations as a function of time. Based on this definition, we find that spiders exhibit transient motor function over time scales of many hours and have a maximum efficiency on the order of 1%, weak compared to other types of molecular motors.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors and present a class of designs for synthetic protein motors that move along a linear DNA track.This presentation will focus on two aspects of the motor design. Firstly the use of self-assembling components whose function is to co-localise the necessary motor functions. We demonstrate that a coiled coil template can be adapted in order to program the self-assembly of three different coiled coils from a solution of six peptides.Secondly the role of linkers between components in controlling dynamics and hence both processivity and power strokes within our motor designs will be discussed. The use of cis-trans isomerization of azobenzene as a mechanism for driving conformational change within the motor will be demonstrated.1. The Tumbleweed: towards a synthetic protein motor, EHC Bromley et al. HFSP J. 3, 204-212 (2009).2. Tuning the performance of an artificial protein motor, NJ Kuwada et al. Phys. Rev. E 84(3) 031922 (2011).3. Designed α-Helical Tectons for Constructing Multicomponent Synthetic Biological Systems EHC Bromley et al. J. Am. Chem. Soc. 131, 928-930 (2009).4. Squaring the circle in peptide assembly: From fibers to discrete Nanostructures by de Novo Design. AL Boyle et al. J. Am. Chem. Soc. 134, 15457-15467 (2012).

The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 μm particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The far-field optical imaging of mitochondria of live cells without the use of any label is demonstrated. It uses a highly sensitive photothermal method and has a resolution comparable to confocal fluorescence setups. The morphological states of mitochondria were followed under different physiological treatments, and the role of cytochrome c was ruled out as the main origin of the photothermal signals. This label free optical method provides a high contrast imaging of live mitochondria and should find many applications in biosciences.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the “Tumbleweed.” This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors.

Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is

Optical tweezers have become an important tool for the manipulation of single biomolecules. However, their application to stretching biopolymers is usually limited to molecules that are several microns in length because conventional optical tweezers manipulate molecules laterally in the focal plane of the microscope objective, a mode in which steric hindrances from the attached microsphere and the surface are substantial. In order to study the properties of short DNA fragments that are typically 1000 bp long, we used optical tweezers in the axial direction to pull microsphere away from the cover glass surface. The microsphere was held in the linear region of the optical potential where the optical force is least sensitive to the bead position. By varying the laser intensity, different stretching forces were applied to the DNA molecule, and the axial position of the tethered microsphere was obtained from its diffraction pattern. The results indicate that the wormlike chain model is still valid for such short DNA fragments.

Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.

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.

We introduce a highly sensitive far-field optical method for imaging individual non-fluorescent nano-objects. This allows for the unprecedented detection of individual gold clusters of only 67 atoms. The spectroscopy of individual nanoclusters is demonstrated.

International audience; We present a novel readout scheme for gold nanoparticle-based DNA microarrays relying on Laser Induced Scattering around a NanoAbsorber (LISNA). It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.

International audience; We present the theoretical and detailed experimental characterizations of Photothermal Heterodyne Imaging. An analytical expression of the photothermal heterodyne signal is derived using the theory of light scattering from a fluctuating medium. The amplitudes of the signals detected in the backward and forward configurations are compared and their frequency dependences are studied. The application of the Photothermal Heterodyne detection technique to the absorption spectroscopy of individual gold nanoparticles is discussed and the detection of small individual silver nanoparticles is demonstrated.

International audience; We introduce a new, highly sensitive, and simple heterodyne optical method for imaging individual nonfluorescent nanoclusters and nanocrystals. A 2 order of magnitude improvement of the signal is achieved compared to previous methods. This allows for the unprecedented detection of individual small absorptive objects such as metallic clusters (of 67 atoms) or nonluminescent semiconductor nanocrystals. The measured signals are in agreement with a calculation based on the scattering field theory from a photothermal-induced modulated index of refraction profile around the nanoparticle.

We describe a simple and straightforward approach for homogeneous and isothermal detection of individual rolling circle replication (RCR) products, which represent individual padlock probe circularization events. The RCR products constitute tens of kilobases long single-stranded tandem repeated copies of the probe sequence, and in solution, they fold into micrometer-sized random coils. The method is based on the local enrichment of fluorescence-labeled probes that hybridize to the coiled RCR products compared to the concentration of free probes in solution. We present a detailed characterization of the fluorescence-labeled products using a highly sensitive and fast microscopy setup. At a 10(4)-fold excess of free label, we were able to detect and follow individual RCR products at a signal-to-background noise ratio of 27. This high signal-to-background noise ratio leaves room for analysis in a simple detection device at higher speeds or at lower labeling ratios.

Molecular spiders are synthetic molecular motors featuring multiple legs such that each leg can interact with a substrate through binding and cleavage. Experimental studies of molecular spiders suggest that the motion of the spider on both a substrate matrix [R. Pei et al., J. Amer. Chem. Soc.128, 12693 (2006)] and a two dimensional substrate track [K. Lund et al, Nature 465, 206 (2010)] is biased towards uncleaved substrates. We first investigated the origin of the spider's biased motion by using Monte Carlo simulations of bipedal spiders on a 1D track based on a realistic chemical kinetic model [L. Samii et al., Phys. Rev. E81, 021106-1 (2010)], and found that substrate cleavage and spider detachment from the track both contribute to biased motion of the spider population. In the work reported here, we extend these studies by investigating how experimental parameters such as number of legs, leg length and substrate cleavage rate can be tuned to optimize the motor properties of the spider. We find that each of these parameters affect properties such as binding time to the track, processivity and speed in different ways. To evaluate further the motor properties of the spider, we calculated the thermodynamic efficiencies of multi-pedal molecular spiders, and found that they are both force and time dependent. By comparing our results with biological molecular motors such as kinesin, we were able to comment on the effect of tight versus loose mechano-chemical coupling on thermodynamic efficiency.

The combined analysis of spectral and lifetime images has the potential to provide more accurate and more detailed information about Förster resonance energy transfer (FRET). We have developed a novel FRET analysis method to analyze images recorded by multispectral lifetime imaging. The new method is based on a phasor approach and facilitates the simultaneous analysis of decay kinetics of donor and acceptor molecules. The method is applicable to both molecules that exhibit a mono-exponential decay and a bi-exponential decay. As an example we show the possibility of extracting the energy transfer efficiency and the fraction of interacting molecules even in the presence of non-interacting molecules. The reliability of the method is investigated by comparing it with conventional FRET-FLIM analyses. We show that, with the same number of detected photons, the spectrally resolved phasor approach provides higher accuracy than other analysis methods; the confidence interval is improved and the FRET efficiency is closer to the real value.

Nonlinear microscopy, which in combination with fluorescence spectroscopy is called nonlinear spectral imaging (NLSI), affords access to specimen morphology and (auto)fluorescence spectra at the same time. Thus, it measures the biochemical composition, while also distinguishing different parts of the tissue. Data interpretation is simplified by a new analysis method, the spectral phasor [1], which results in a robust, quick, and semi-blind spectral unmixing of fluorescent species.NLSI and the spectral phasor are a very user-friendly technique and have the potential to address a broad range of microbiological questions. We introduce them as a novel minimum-invasive technique to monitor the state of "fungal cells" (hyphae). Fungi, both used as consumables and organisms to produce industrial and pharmaceutical compounds, require stringent quality control during their growth. To this end, and as one possible application of NLSI and the spectral phasor, we present their use in monitoring the quality and freshness of white button mushrooms.[1] F. Fereidouni, A.N. Bader, and H.C. Gerritsen, Optics Express, 20,12729-12741 (2012).RGB-representation of a nonlinear spectral image of the white button mushroom (A. bisporus, left) and the corresponding phasor.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Based on a recent approach to understanding protein-based molecular motors (1), we present computer simulations of a novel nanomotor concept dubbed the Synthetic Kinesin Analog Motor Protein (SKAMP). SKAMP is a purely diffusive linear motor consisting of four ligand gated DNA-binding (repressor) proteins of two types, A and B, linked by three rigid coiled-coils (‘rods’) to form a complex A1-A2-B2-B1, the length of the central rod being shorter than the outer two. Directional stepping along a periodic DNA track is maintained by a temporally periodic external chemical supply. Due to the shorter central rod, SKAMP makes use of a mechanism analogous to that used by kinesin which involves docking of a neck linker onto a motor domain. This mechanism allows kinesin to reduce its diffusional search time for a binding site. We use coarse-grained Langevin Dynamics simulations in the overdamped limit to study the detailed motion of SKAMP when subject to a load force, SKAMP acting as a shuttle and the increase of SKAMP's performance by external feedback.1. Bromley et al. HFSP Journal 3 204-213 (2009).View Large Image | View Hi-Res Image | Download PowerPoint Slide

We report the observation of single-molecule colocalization and quantitative fluorescence resonant energy transfer by simultaneously imaging the emission and polarization characteristics of two colocalized fluorophores using a simple optical design. The methodology was tested using the ligand-receptor system streptavidin, fluorescence labeled with the dye Cy5, and biotin labeled with tetramethylrhodamine. Discrimination of the two dyes permitted the observation of single-pair fluorescence resonant energy transfer with an efficiency of 89%. The multidimensional character of our fluorescence microscopy combined with the robustness of our design provides a simple method suitable to study biomolecular interactions on the single molecule level.

A method, is presented for blind unmixing spectrally resolved fluorescence lifetime images. The method is based on the combined analysis of spectral and lifetime phasors and allows unmixing of up to three components without any prior knowledge. Fractional intensities, spectra and decay curves of the individual components can be extracted with this new technique. The reliability and sensitivity are investigated and the possibility of extending the method to unmix more components is discussed. The method is evaluated on mixtures of fluorescent dyes and labeled cells.

In a bottom up approach to understanding molecular motors, a synthetic protein-based molecular motor, the “tumbleweed”, is being designed and constructed [1]. This design uses three ligand dependent DNA repressor proteins to rectify diffusive motion of the construct along a DNA track. These proteins are MetJQ44K, TrpR, and DtxR. To predict the behavior of this artificial motor one needs to understand the binding and unbinding kinetics of the repressor proteins at a single-molecule level. An assay, similar to tethered particle motions assays [2], is used to measure the unbinding rates of these three DNA repressor proteins. In this assay the repressor is immobilized to a surface in a microchamber. Long DNA with the correct recognition sequence for one of the repressors is attached to a streptavidin-coated microsphere. As the DNA-microsphere construct diffuses through the microchamber it will sometimes bind to the repressor protein. Using brightfield microscopy and a CCD camera the diffusive motion of the microsphere can be characterized and bound and unbound states can be differentiated. On the order of ten microspheres can be easily visualized at one time allowing single-molecule measurements to be done in parallel. The resulting kinetic measurements are compared to bulk binding kinetics measured in a QCM device.[1] EHC Bromley et al. 2009. The Tumbleweed: Towards a synthetic protein motor. HFSP Journal. 3:204-212.[2] F Vanzi et al. 2006. Lac Repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion. Nuc. Acids. Res. 34:3409-3420.

Protein-mediated DNA looping is a ubiquitous motif in transcriptional control schemes. Formation of these loops is driven by thermal fluctuations of the substrate DNA, which in turn are known to be exquisitely sensitive to mechanical constraints on the DNA. Because DNA in vivo is subject to a complex micromechanical environment, it is intriguing to study the effect of mechanical tension in the substrate DNA on the formation of these regulatory loops to investigate the role of mechanics in controlling gene regulation. For this purpose, we measured the formation and breakdown rates of lac repressor-mediated DNA loops under tension using constant-force axial optical tweezers.We observed that an incremental force of less than 100 femtonewtons is sufficient to reduce loop formation rate about sevenfold in a construct with an inter-operator spacing of 305 bp. This result suggests the possibility of mechanical pathways to control gene expression with forces that are two orders of magnitude lower than other typical intracellular forces acting on DNA, such as the forces exerted by RNA polymerase and molecular motors. Moreover, we developed a model that quantifies the relation between the force sensitivity of the loop formation rate and the angle between the incoming and outgoing DNA strand in the loop as a way to infer loop topology from our micromechanical measurements. We conclude that the LacI-mediated DNA loop prefers an anti-parallel loop topology over a parallel conformation.

A method that combines fluorescence imaging and spectroscopy of single molecules at room temperature is presented. This approach allows us to identify a number of imaged molecules unequivocally by simultaneously recording their fluorescence emission spectra. Furthermore, their spectral characteristics not only allow us to separate different fluorescent labels quantitatively and qualitatively but also provide information on the microenvironment of the molecules. This new method was successfully tested on a system of yellow-green and red fluorescent 20-nm latex beads, and its usefulness in studies of biological systems was illustrated for a preparation of combined binary ratio labeling-fluorescence in situ hybridization-stained mouse chromosomes.

International audience; In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.

L-type Ca 2+ channels are an important means by which a cell regulates the Ca 2+ influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca 2+ channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca 2+ channels tend to form larger aggregates which are mobile in the plasma membrane.

We present a newly developed photothermal imaging technique to perform absorption spectroscopy of individual nano-objects. Intrinsic size effects in the surface plasmon resonance of gold nanoparticles and CdSe/ZnS nanocrystals in the multiexcitonic regime are studied.

Though the biological function of many natural molecular motors is fairly well established, many structure-function details responsible for motor performance remain vague or unknown completely. Recently, we have undertaken a new bottom-up approach to understanding biological molecular motors by designing and building an artificial, protein-based molecular motor dubbed the Tumbleweed (TW). The TW is a purely diffusive motor construct consisting of three DNA-binding proteins attached to a designed, protein-based central hub, where directional stepping along a DNA track is maintained by a temporally periodic external chemical supply. To better understand important design and performance characteristics of the TW, coarse-grained Langevin Dynamics (LD) simulations and numerical solutions to the Master Equation (ME) were carried out. The LD approach, which is a single motor simulation, is particularly suitable for exploring the diffusional behavior of the system, where the ME approach, which models an ensemble of motor states, is best suited for statistically exploring the parameter space of the system and the interaction of processes at different time scales. We present results from these two theoretical approaches that illuminate not only important design and experimental considerations, such as motor geometry and track spacing, but also produce unexpected diffusional behavior. Of particular interest is that the addition of certain internal symmetric potentials can increase motor performance. For example, the addition of a non-specific binding potential, symmetric about the DNA track, can double motor speed by replacing some of the 3D diffusional search by a relatively fast 1D diffusional slide along the DNA. This, and other symmetric potential inputs that increase motor performance by subtly amplifying asymmetries in the system, are not only fundamentally interesting but also may be applicable to any molecular motor that incorporates a diffusional search in its stepping cycle.

Molecular spiders are synthetic molecular motors featuring multiple legs that each can interact with a substrate through binding and cleavage. Experimental studies suggest the motion of the spider in a matrix is biased towards uncleaved substrates, and that spider properties such as processivity can be altered by changing the binding strength of the legs to substrate [R. Pei et al., J. Amer. Chem. Soc.128, 12693 (2006)]. We investigate the origin of biased motion and molecular motor properties of bipedal spiders using Monte Carlo simulations. Our simulations combine a realistic chemical kinetic model, hand-over-hand (HOH) or inchworm (IW) modes of stepping, and the use of a 1D track. We find that substrate cleavage and spider detachment from the track are both contributing mechanisms to population bias but are not necessary for biased motion on an asymmetric track. We investigate the contributions of stepping mechanism to speed, randomness parameter, processivity, coupling and efficiency, and comment on how these molecular motor properties can be altered by changing experimentally tunable kinetic parameters. We then consider the more general case where steps can occur by any mechanism, subject to steric constraints. We compare these results with the above for bipedal spiders and then simulate quadrupedal spiders to investigate the effect of leg number on motor performance.