Josef Lazar

SRp38 Regulates Alternative Splicing and Is Required for Ca2+ Handling in the Embryonic Heart

SRp38 is an atypical SR protein splicing regulator. To define the functions of SRp38 in vivo, we generated SRp38 null mice. The majority of homozygous mutants survived only until E15.5 and displayed multiple cardiac defects. Evaluation of gene expression profiles in the SRp38(-/-) embryonic heart revealed a defect in processing of the pre-mRNA encoding cardiac triadin, a protein that functions in regulation of Ca(2+) release from the sarcoplasmic reticulum during excitation-contraction coupling. This defect resulted in significantly reduced levels of triadin, as well as those of the interacting protein calsequestrin 2. Purified SRp38 was shown to bind specifically to the regulated exon and to modulate triadin splicing in vitro. Extending these results, isolated SRp38(-/-) embryonic cardiomyocytes displayed defects in Ca(2+) handling compared with wild-type controls. Taken together, our results demonstrate that SRp38 regulates cardiac-specific alternative splicing of triadin pre-mRNA and, reflecting this, is essential for proper Ca(2+) handling during embryonic heart development.

Two-photon polarization microscopy reveals protein structure and function

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.

Absolute frequency shifts of iodine cells for laser stabilization

We present an investigation of iodine cell purity and influence of contaminations upon frequency shifts of iodine-stabilized frequency-doubled Nd : YAG lasers. The study combines measurements of laser-induced fluorescence and evaluation through the Stern–Volmer formula, with direct measurement of frequency shifts referenced by means of an optical comb to a radiofrequency clock etalon. These indirect and direct approaches are compared and provide feedback on the cell manufacturing procedure. Significant improvement of the apparatus for the measurement of induced fluorescence is reported, leading to better repeatability of the results. The ultimate precision that can be achieved in measurements of the absolute frequency of a stabilized laser is discussed in terms of the cell quality. (Some figures in this article are in colour only in the electronic version)

Conversion of stability of femtosecond mode-locked laser to optical cavity length

In this contribution we propose a scheme for a generation of precise displacements through conversion of relative stability of components of a femtosecond laser into the length of a Fabry-Perot cavity. The spacing of mirrors of a Fabry-Perot interferometer represents a mechanical length standard referenced to stable optical frequency of a femtosecond mode-locked laser. With the help of a highly selective optical filter, it is possible to get only a few discrete spectral components. By tuning and locking the Fabry-Perot cavity to a selected single component it is possible to get a mechanical length standard with the uncertainty of the repetition frequency of the femtosecond laser. To verify the method, an auxiliary single-frequency laser is locked to the resonance mode of the cavity and simultaneously it is optically mixed with an independent optical frequency standard He-Ne-I2. The stability of the beat-frequency between these 2 lasers represents the stability of the Fabry-Perot cavity length. The stability recording evaluated through Allan variances for one hour of operation is presented. The pilot experimental setup is able to generate the length standard in the order of 0.01 nm for 20 min of integration time.

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 ArcLights 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.

Local probe microscopy with interferometric monitoring of the stage nanopositioning

We present a system of positioning and interferometric monitoring of a sample position for measurements and calibration in the nanoscale in metrology. The positioning is based on a three-axis stage which allows replacing scanning by the probe of an atomic force microscope with a system with full interferometric displacement measurement. A stage with 200 µm × 200 µm of horizontal travel extends also the microscope range. The stage allows positioning with sub-nanometer resolution in all three axes under a closed loop control with position detection via capacitive sensors. Interferometric system monitoring all six degrees of freedom of the stage ensures full metrological traceability of the positioning to the fundamental etalon of length and improves resolution and overall precision of the displacement monitoring.

Small displacement measurements with subatomic resolution by beat frequency measurements

In this paper a novel method for high-resolution measurement of displacements with sub-atomic resolution is described. With this method, a length change of an optical resonator is directly transformed to a radio-frequency signal. A tunable He?Ne laser is locked to a mode of the resonator using a digital signal processing technique. Heterodyne mixing of this locked laser with an iodine-stabilized He?Ne laser converts the frequency of the laser locked to the cavity into the radio-frequency region. A HF counter measures the beat frequency from which the displacement can be derived directly. This method delivers inherent linearity and sub-nanometre resolution of the displacement over a range of several micrometres. An example of the capabilities of this system is given in this paper, where it is used for checking periodic deviations of a laser interferometer system. Emphasis is put on the construction of the optical resonator, on how its narrow resonance line-width is achieved, and how the required mechanical stability is achieved. The measurement range and the scale linearity are discussed in detail. Possible applications of this method are the calibration of nano-position systems based on PZT transducers, as well as inductive and capacitive sensors.

Laser diode current controller with a high level of protection against electromagnetic interference

We present a current controller which satisfies the highest protection criteria of semiconductor lasers notorious for their great sensitivity to damage caused by induced electromagnetic interference. The core current source is supplied by linear isolating converter providing ripple free voltage. It is galvanically isolated, double shielded and current sense as well as current modulation are coupled via linear optocouplers. The current controller in this configuration makes safe operation of semiconductor lasers in laboratory conditions possible.

Accurate Determination of the Orientational Distribution of a Fluorescent Molecule in a Phospholipid Membrane

Orientation of lipophilic dye molecules within a biological membrane can report on the molecular environment, i.e., the physical and chemical properties of the surrounding membrane. This fact, however, remains under-utilized, largely because of our limited quantitative knowledge of molecular orientational distributions and the fact that robust techniques allowing experimental observation of molecular orientations of dyes in biological membranes are only being developed. In order to begin filling this lack of knowledge and to develop appropriate tools, we have investigated the membrane orientational distribution of the 3-hydroxyflavone-based membrane dye F2N12S. Results of our single- and two-photon polarization microscopy observations of linear dichroism of F2N12S-labeled giant unilamellar vesicles are consistent with a Gaussian-like orientational distribution of the transition dipole moment of the dye, with a mean tilt angle of 53.2 ± 0.1° with respect to the bilayer normal and a standard deviation of 13.3 ± 0.6°. Independently, by combining quantum chemical calculations and molecular dynamics simulations, we obtained very similar values; a mean tilt angle of 48 ± 4° and a standard deviation of 13 ± 2°. The good agreement between the experimentally and computationally obtained values cross-validates both approaches and gives confidence to the results obtained. The results open a door to robust quantitative determinations of orientational distributions of fluorescent molecules (ranging from simple synthetic dyes to fluorescent proteins attached to membrane proteins) associated with lipid membranes. Such determinations enable rational development of a novel class of sensitive fluorescent optical probes, reporting on cellular events through changes in linear dichroism.

Highly coherent tunable semiconductor lasers in metrology of length

Laser diodes became the most widespread lasers and now are available in a broad spectrum of wavelengths ranging from infrared to the visible region. The low power ones mainly those with the quantum well structure and gain or index guided configuration perform a narrow linewidth and soon became a favourite tool for interferometry and spectroscopy. The need for continuous tuning range led to the development Extended Cavity Laser systems (ECL) and Vertical Cavity Surface Emitting Lasers (VCSEL). Both systems seem to be promising laser sources for design of optical frequency standards or interferometric distance measurement devices. We present design of these laser systems and their applications in metrology of length.