Dynamics of transition dipole moment orientation in representative fluorescent proteins.

Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies (r0) consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.

Design and Characterisation of an Optical Fibre Dosimeter Based on Silica Optical Fibre and Scintillation Crystal.

In nuclear power plants, particle accelerators, and other nuclear facilities, measuring the level of ionising gamma radiation is critical for the safety and management of the operation and the environment's protection. However, in many cases, it is impossible to monitor ionising radiation directly at the required location continuously. This is typically either due to the lack of space to accommodate the entire dosimeter or in environments with high ionising radiation activity, electromagnetic radiation, and temperature, which significantly shorten electronics' lifetime. To allow for radiation measurement in such scenarios, we designed a fibre optic dosimeter that introduces an optical fibre link to deliver the scintillation radiation between the ionising radiation sensor and the detectors. The sensors can thus be placed in space-constrained and electronically hostile locations. We used silica optical fibres that withstand high radiation doses, high temperatures, and electromagnetic interference. We use a single photon counter and a photomultiplier to detect the transmitted scintillation radiation. We have shown that selected optical fibres, combined with different scintillation materials, are suitable for measuring gamma radiation levels in hundreds of kBq. We present the architecture of the dosimeter and its experimental characterisation with several combinations of optical fibres, detectors, and scintillation crystals.

Coherent fibre link for synchronization of delocalized atomic clocks.

Challenging experiments for tests in fundamental physics require highly coherent optical frequency references with suppressed phase noise from hundreds of kHz down to µHz of Fourier frequencies. It can be achieved by remote synchronization of many frequency references interconnected by stabilized optical fibre links. Here we describe the path to realize a delocalized optical frequency reference for spectroscopy of the isomeric state of the nucleus of Thorium-229 atom. This is a prerequisite for the realization of the next generation of an optical clock - the nuclear clock. We present the established 235 km long phase-coherent stabilized cross-border fibre link connecting two delocalized metrology laboratories in Brno and Vienna operating highly-coherent lasers disciplined by active Hydrogen masers through optical frequency combs. A significant part (up to tens of km) of the optical fibre is passing urban combined collectors with a non-negligible level of acoustic interference and temperature changes, which results in a power spectral density of phase noise over 105 rad2· Hz-1. Therefore, we deploy a digital signal processing technique to suppress the fibre phase noise over a wide dynamic range of phase fluctuations. To demonstrate the functionality of the link, we measured the phase noise power spectral density of a remote beat note between two independent lasers, locked to high-finesse stable resonators. Using optical frequency combs at both ends of the link, a long-term fractional frequency stability in the order of 10-15 between local active Hydrogen masers was measured as well. Thanks to this technique, we have achieved reliable operation of the phase-coherent fibre link with fractional stability of 7 × 10-18 in 103 s.

Absolute frequencies of H13C14N hydrogen cyanide transitions in the 1.5-µm region with the saturated spectroscopy and a sub-kHz scanning laser.

The wide span and high density of lines in its rovibrational spectrum render hydrogen cyanide a useful spectroscopic media for referencing absolute frequencies of lasers in optical communication and dimensional metrology. We determined, for the first time to the best of our knowledge, the molecular transitions' center frequencies of the H13C14N isotope in the range from 1526 nm to 1566 nm with 1.3 × 10-10 fractional uncertainty. We investigated the molecular transitions with a highly coherent and widely tunable scanning laser that was precisely referenced to a hydrogen maser through an optical frequency comb. We demonstrated an approach to stabilize the operational conditions needed to maintain the constantly low pressure of the hydrogen cyanide to carry out the saturated spectroscopy with the third-harmonic synchronous demodulation. We demonstrated approximately a forty-fold improvement in the line centers' resolution compared to the previous result.

Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools.

Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.

Optical sensors of heterotrimeric G protein signaling.

Heterotrimeric G proteins are central mediators of cellular signal transduction. They receive, process, and transduce signals from G protein-coupled receptors to downstream effectors. Since their discovery, a number of optical sensors of G protein localisation and function have been developed and applied in living systems. In this minireview, we provide an overview of existing G protein-based sensors and the experimental approaches they utilise, with emphasis on live-cell imaging techniques. We outline recent advances, as well as identify current challenges and likely future directions in the field of G protein sensor development.

Directionality of light absorption and emission in representative fluorescent proteins.

Fluorescent molecules are like antennas: The rate at which they absorb light depends on their orientation with respect to the incoming light wave, and the apparent intensity of their emission depends on their orientation with respect to the observer. However, the directions along which the most important fluorescent molecules in biology, fluorescent proteins (FPs), absorb and emit light are generally not known. Our optical and X-ray investigations of FP crystals have now allowed us to determine the molecular orientations of the excitation and emission transition dipole moments in the FPs mTurquoise2, eGFP, and mCherry, and the photoconvertible FP mEos4b. Our results will allow using FP directionality in studies of molecular and biological processes, but also in development of novel bioengineering and bioelectronics applications.

Comparison of three focus sensors for optical topography measurement of rough surfaces.

The study compares three variants of focus sensors designed for the optical topography measurement of rough surface specimens with submicron accuracy. We present a theoretical analysis of the focus sensor principles and the experimental measurements with a single point laser probe. A low coherent illumination beam was provided by a monochromatic laser source and a rotating diffuser, which reduced the speckles generated by the rough surface. The reflected beam was modulated by three specific optical elements (axicon, double wedge prism, four spherical lenses) realized by a spatial light modulator. A digital camera detected the output intensity patterns that were evaluated by the intensity centroid method. The results showed a good coincidence of the surface profiles obtained by the three sensor variants with the root-mean-square deviations below one micron. We discuss the results obtained for several specimens with various surface roughness and compare the differences between the three focus sensor variants.

The activity of Saccharomyces cerevisiae Na+, K+/H+ antiporter Nha1 is negatively regulated by 14-3-3 protein binding at serine 481.

Na+/H+ antiporters are involved in ensuring optimal intracellular concentrations of alkali-metal cations and protons in most organisms. In Saccharomyces cerevisiae, the plasma-membrane Na+, K+/H+ antiporter Nha1 mediates Na+ and K+ efflux, which is important for cell growth in the presence of salts. Nha1 belongs among housekeeping proteins and, due to its ability to export K+, it has many physiological functions. The Nha1 transport activity is regulated through its long, hydrophilic and unstructured C-terminus (554 of 985 aa). Although Nha1 has been previously shown to interact with the yeast 14-3-3 isoform (Bmh2), the binding site remains unknown. In this work, we identified the residues through which Nha1 interacts with the 14-3-3 protein. Biophysical characterization of the interaction between the C-terminal polypeptide of Nha1 and Bmh proteins in vitro revealed that the 14-3-3 protein binds to phosphorylated Ser481 of Nha1, and the crystal structure of the phosphopeptide containing Ser481 bound to Bmh1 provided the structural basis of this interaction. Our data indicate that 14-3-3 binding induces a disorder-to-order transition of the C-terminus of Nha1, and in vivo experiments showed that the mutation of Ser481 to Ala significantly increases cation efflux activity via Nha1, which renders cells sensitive to low K+ concentrations. Hence, 14-3-3 binding is apparently essential for the negative regulation of Nha1 activity, which should be low under standard growth conditions, when low amounts of toxic salts are present and yeast cells need to accumulate high amounts of K+.

Influence of coating technology and thermal annealing on the optical performance of AR coatings in iodine-filled absorption cells.

In this contribution, we investigate the properties of antireflective coatings on iodine-filled absorption cell windows. These coatings are subject to high temperatures during the cell production process and are in direct contact with the absorption medium, which influences their optical performance. We tested the thermal resistance of TiO2- and Ta2O5- based coatings produced using conventional electron beam evaporation (e-beam) and ion-assisted deposition (PIAD). We prepared a set of iodine-filled absorption cells that were used to test the coatings' resistance to iodine vapors. We show that the choice of coating materials, coating methods, and a well-chosen bakeout procedure can mitigate any unwanted effects, such as temperature-induced spectral shifts and optical losses inhomogeneities or settling of the absorption medium in the coating.

The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

The Gi/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of Gi/o-GPCR coupling and the high rate of Gi/o signal transduction have been hypothesized to be enabled by the existence of stable associates between Gi/o proteins and their cognate GPCRs in the inactive state (Gi/o-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gαi1 subunits labeled with fluorescent proteins and four GPCRs: the α2A-adrenergic receptor, GABAB, cannabinoid receptor type 1 (CB1R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gαi1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gαi1 subunits and α2A-adrenergic receptor, GABAB, or dopamine receptor type 2 receptors did not reveal any interaction between the Gi1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB1R exhibited an interaction with the Gi1 protein. Further experiments revealed that this interaction is caused solely by CB1R basal activity; no preassembly between CB1R and the Gi1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active Gi1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and Gi1 (basal coupling). These findings suggest that Gi1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs.

Iodine Absorption Cells Purity Testing.

This article deals with the evaluation of the chemical purity of iodine-filled absorption cells and the optical frequency references used for the frequency locking of laser standards. We summarize the recent trends and progress in absorption cell technology and we focus on methods for iodine cell purity testing. We compare two independent experimental systems based on the laser-induced fluorescence method, showing an improvement of measurement uncertainty by introducing a compensation system reducing unwanted influences. We show the advantages of this technique, which is relatively simple and does not require extensive hardware equipment. As an alternative to the traditionally used methods we propose an approach of hyperfine transitions' spectral linewidth measurement. The key characteristic of this method is demonstrated on a set of testing iodine cells. The relationship between laser-induced fluorescence and transition linewidth methods will be presented as well as a summary of the advantages and disadvantages of the proposed technique (in comparison with traditional measurement approaches).

Noise Suppression on the Tunable Laser for Precise Cavity Length Displacement Measurement.

The absolute distance between the mirrors of a Fabry-Perot cavity with a spacer from an ultra low expansion material was measured by an ultra wide tunable laser diode. The DFB laser diode working at 1542 nm with 1.5 MHz linewidth and 2 nm tuning range has been suppressed with an unbalanced heterodyne fiber interferometer. The frequency noise of laser has been suppressed by 40 dB across the Fourier frequency range 30-300 Hz and by 20 dB up to 4 kHz and the linewidth of the laser below 300 kHz. The relative resolution of the measurement was 10 - 9 that corresponds to 0.3 nm (sub-nm) for 0.178 m long cavity with ability of displacement measurement of 0.5 mm.

Nonlinear Optical Properties of Fluorescent Dyes Allow for Accurate Determination of Their Molecular Orientations in Phospholipid Membranes.

Several methods based on single- and two-photon fluorescence detected linear dichroism have recently been used to determine the orientational distributions of fluorescent dyes in lipid membranes. However, these determinations relied on simplified descriptions of nonlinear anisotropic properties of the dye molecules, using a transition dipole-moment-like vector instead of an absorptivity tensor. To investigate the validity of the vector approximation, we have now carried out a combination of computer simulations and polarization microscopy experiments on two representative fluorescent dyes (DiI and F2N12S) embedded in aqueous phosphatidylcholine bilayers. Our results indicate that a simplified vector-like treatment of the two-photon transition tensor is applicable for molecular geometries sampled in the membrane at ambient conditions. Furthermore, our results allow evaluation of several distinct polarization microscopy techniques. In combination, our results point to a robust and accurate experimental and computational treatment of orientational distributions of DiI, F2N12S, and related dyes (including Cy3, Cy5, and others), with implications to monitoring physiologically relevant processes in cellular membranes in a novel way.

Self-referenced interferometer for cylindrical surfaces.

We present a new interferometric method for shape measurement of hollow cylindrical tubes. We propose a simple and robust self-referenced interferometer where the reference and object waves are represented by the central and peripheral parts, respectively, of the conical wave generated by a single axicon lens. The interferogram detected by a digital camera is characterized by a closed-fringe pattern with a circular carrier. The interference phase is demodulated using spatial synchronous detection. The capabilities of the interferometer are experimentally tested for various hollow cylindrical tubes with lengths up to 600 mm.

Spectral properties of molecular iodine in absorption cells filled to specified saturation pressure.

We present the results of measurement and evaluation of spectral properties of iodine absorption cells filled at certain saturation pressure. A set of cells made of borosilicate glass instead of common fused silica was tested for their spectral properties in greater detail with special care for the long-term development of the absorption media purity. The results were compared with standard fused silica cells and the high quality of iodine was verified. A measurement method based on an approach relying on measurement of linewidth of the hyperfine transitions is proposed as a novel technique for iodine cell absorption media purity evaluation. A potential application in laser metrology of length is also discussed.

Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

Two-stage system based on a software-defined radio for stabilizing of optical frequency combs in long-term experiments.

A passive optical resonator is a special sensor used for measurement of lengths on the nanometer and sub-nanometer scale. A stabilized optical frequency comb can provide an ultimate reference for measuring the wavelength of a tunable laser locked to the optical resonator. If we lock the repetition and offset frequencies of the comb to a high-grade radiofrequency (RF) oscillator its relative frequency stability is transferred from the RF to the optical frequency domain. Experiments in the field of precise length metrology of low-expansion materials are usually of long-term nature so it is required that the optical frequency comb stay in operation for an extended period of time. The optoelectronic closed-loop systems used for stabilization of combs are usually based on traditional analog electronic circuits processing signals from photodetectors. From an experimental point of view, these setups are very complicated and sensitive to ambient conditions, especially in the optical part, therefore maintaining long-time operation is not easy. The research presented in this paper deals with a novel approach based on digital signal processing and a software-defined radio. We describe digital signal processing algorithms intended for keeping the femtosecond optical comb in a long-time stable operation. This need arose during specialized experiments involving measurements of optical frequencies of tunable continuous-wave lasers. The resulting system is capable of keeping the comb in lock for an extensive period of time (8 days or more) with the relative stability better than 1.6 × 10(-11).

Short-range six-axis interferometer controlled positioning for scanning probe microscopy.

We present a design of a nanometrology measuring setup which is a part of the national standard instrumentation for nanometrology operated by the Czech Metrology Institute (CMI) in Brno, Czech Republic. The system employs a full six-axis interferometric position measurement of the sample holder consisting of six independent interferometers. Here we report on description of alignment issues and accurate adjustment of orthogonality of the measuring axes. Consequently, suppression of cosine errors and reduction of sensitivity to Abbe offset is achieved through full control in all six degrees of freedom. Due to the geometric configuration including a wide basis of the two units measuring in y-direction and the three measuring in z-direction the angle resolution of the whole setup is minimize to tens of nanoradians. Moreover, the servo-control of all six degrees of freedom allows to keep guidance errors below 100 nrad. This small range system is based on a commercial nanopositioning stage driven by piezoelectric transducers with the range (200 × 200 × 10) µm. Thermally compensated miniature interferometric units with fiber-optic light delivery and integrated homodyne detection system were developed especially for this system and serve as sensors for othogonality alignment.

Dissociated GαGTP and Gßγ protein subunits are the major activated form of heterotrimeric Gi/o proteins.

Although most heterotrimeric G proteins are thought to dissociate into Gα and Gßγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.