Zygmunt J. Jakubek

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers.

The effect of enzymatic generation of ceramide on phase separated bilayers with a mixture of co-existing fluid and liquid-ordered phases has been examined using a combination of atomic force microscopy (AFM) and fluorescence imaging. Supported lipid bilayers prepared from a DOPC/sphingomyelin/cholesterol mixture were imaged prior to, during and after incubation with sphingomyelinase by total internal reflection fluorescence (TIRF) microscopy. Enzyme treatment resulted in the growth of large dye-excluded regions. The growth kinetics for these patches are consistent with activity of a variable number of enzyme molecules in different regions of the bilayer. Correlated AFM and fluorescence imaging shows that some of the large dye-excluded patches form around the original liquid-ordered domains, which become heterogeneous in height with many raised ceramide-rich regions around their periphery. However, some of the dye-excluded patches correspond to areas of the bilayer where the initial domains have largely or partially disappeared. The dye-excluded patches observed by fluorescence are shown to be areas of increased adhesion in lateral deflection AFM images and are postulated to form by incorporation of both cholesterol and ceramide in the original fluid phase and to vary in composition throughout the bilayer. This is evident from the observation that the dye-excluded areas are all detected as areas of increased friction, but do not always show a distinct height difference in topographic images. These results highlight the utility of a multi-modal imaging approach for understanding the complex membrane restructuring that occurs upon enzymatic generation of ceramide.

A Dual Emissive BODIPY Dye and Its Use in Functionalizing Highly Monodispersed PbS Nanoparticles

BODIPY (boron-dipyrromethene) dyes and derivatives are well-known to be very effective in light-harvesting and energy-transfer processes, owing to their high fluorescence quantum yields, large molar absorption coefficients, relatively long excited-state lifetimes, and excellent photochemical stability. Thus, they have frequently been used in lightharvesting molecules, as dye sensitizers, and as probes and labels for biomolecules. One important class of materials for various optoelectronic applications, including solar cells, is the class of semiconductor nanoparticles (NPs). The optical and energetic properties of these nanoparticles can be tuned by varying their shape, size, or surface ligands. Lead sulfide NPs are particularly attractive among NPs owing to their narrow band gap, large exciton Bohr radii, and their absorption and emission in the near-IR region. Several recent reports have shown that PbS NPs are very promising materials for achieving high-performance photovoltaic devices. 5, 6] The most commonly used surface ligand for PbS NPs is oleic acid, which can effectively protect the NPs from oxidation and can facilitate their dispersion in organic solvents. However, because they lack any interesting photophysical properties, oleic acid capping ligands do not engage in any electronic communication or interactions with the NPs and thus have little influence on the properties of the NPs and insulate NPs from each other and the surrounding medium. New surface ligands that can communicate electronically with PbS NPs and enhance their performance in optoelectronic devices are therefore in demand. Based on this consideration and the very attractive photophysical properties of BODIPY dyes, we initiated the investigation of new BODIPY dyes as potential new surface ligands for PbS NPs. Herein, we report the synthesis and photophysical properties of a new BODIPY dye (BDY) and its use in PbS NPs functionalization. The procedure used to synthesize BDY is illustrated in Scheme 1. The bromophenyl-BODIPY starting material 1 was synthesized by using a modified literature procedure.

The SiP molecule: The first observation and spectroscopic characterization

SiP molecules have been produced in a molecular free jet apparatus by laser vaporizing a silicon rod in the presence of He doped with phosphine (PH3) gas. Excitation spectra have been observed in the 21200–22350 cm–1 range by monitoring laser induced fluorescence. Dispersed fluorescence spectra have been recorded out of the upper states of the excitation bands. The ground electronic state is X 2Πi with the spin–orbit splitting A=−186.774(11) cm−1, the harmonic wavenumber ωe=615.7(6) cm−1, and the equilibrium internuclear distance re=2.0775(17) A. Two excited electronic states, A 2Σ+ and B 2Σ+, are observed at T0=427.4(5) and 21317.1(1) cm−1, respectively. The harmonic wavenumbers, ωe, and the equilibrium internuclear distances, re, are 680(2) cm−1 and 1.9658(13) A, respectively, for the A 2Σ+ state and 455(1) cm−1 and 2.1278(8) A, respectively, for the B 2Σ+ state. For the X 2Πi, A 2Σ+, and B 2Σ+ electronic states, RKR potentials have been generated. Franck–Condon factors for the B–X, B–A, and A–X systems...

The excited electronic states and the ionization potential of the AlND3 complex

The AlND3 complex was studied in a molecular beam by resonant one-color two-photon and two-color two-photon photoionization techniques with a resolution of 0.3 cm−1. Six progressions of bands were observed in the 18 100–26 200 cm−1 (381–552 nm) region. One progression (ωe=316±0.8 cm−1) was assigned to the Al–ND3 stretch (ν3′) in the B2A1 state correlating with the 3s 2S term of Al. The origin of the B2A1–X2E1/2 system was found at 18 532.5±0.7 cm−1. The second (ωe=428±2 cm−1) and third (ωe=594±12 cm−1) progressions were assigned to the Al–ND3 stretch (ν3′) and the bend (ν6′) in the C2E state correlating with the 3d 2D term of Al. The origin of the C2E–X2E1/2 system was found at 21 185±5 cm−1. Assignments of the other three progressions with origins and harmonic wave numbers of 22 667±2 and 358±2 cm−1; 24 382±10 and 432±10 cm−1; and ∼21 871 and ∼1050 cm−1 remain uncertain. The spin-orbit splitting of the X2E ground state was determined to be 55.8±0.7 cm−1. Several Rydberg series converging to the v3...

High resolution molecular beam study of the origin band of the B̃ 2Σ+–X̃ 2Σ+ system of yttrium imide

The (0,0,0)–(0,0,0) band of the B 2Σ+–X 2Σ+ system of three isotopomers of yttrium imide (Y14NH, Y15NH, and Y14ND) has been studied by laser-induced fluorescence in a molecular beam apparatus. Rotational, fine, and nuclear magnetic hyperfine structures have been resolved and analyzed. The B 2Σ+(0,0,0) state of Y14NH, Y14ND, and Y15NH is severely perturbed below J=30.5 by eight, three, and two vibronic states, respectively. Although, the nature of these perturbing states can only be speculated upon, their symmetries are either 2Σ or 2Π, and this has made it possible to deperturb the B 2Σ+ state successfully. The spectra can be reproduced within 140 MHz (0.0047 cm−1). The analyses confirm that the molecule is linear in both states with the nuclear arrangement Y–N–H. The bond lengths in the ground X 2Σ+ state and the B 2Σ+ state have been derived to be rY–N=1.877 57(13) A, rN–H=1.0067(10) A, and rY–N=1.8839(43) A, rN–H=1.242(30) A, respectively. The results are compared with the values of ab initio cal...

Studies of a viral suppressor of RNA silencing p19-CFP fusion protein: A FRET-based probe for sensing double-stranded fluorophore tagged small RNAs

Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) p19-CFP fusion protein and Cy3-labeled siRNA. The CIRV p19 fusion protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET. [corrected]

Electronic structure of YN: the X1Σ+ ground state and the a3Σ+ and A1Σ+ excited states

Abstract YN molecules have been produced in a pulsed free jet apparatus by laser vaporizing yttrium metal in the presence of He doped with NH3. Laser induced dispersed fluorescence spectra have been studied. Previously unobserved low-energy excited state, a 3 Σ + , has been discovered. RKR potential curves of the X 1 Σ + ground state as well as of the A 1 Σ + and a 3 Σ + excited states have been determined. Harmonic wavenumbers have been measured to be equal to: 633.2(10), 1007.4(25), and 805.12(63) cm −1 for the X 1 Σ + , A 1 Σ + , and a 3 Σ + states, respectively.

Quantitative Energy Dispersive X-ray Microanalysis of Electron Beam-Sensitive Alloyed Nanoparticles

An energy dispersive X-ray spectrometry (EDXS) method is developed to evaluate the composition of alloyed nanoparticles (NPs) where one of the alloying elements is removed under the electron beam during microanalysis with a transmission electron microscope (TEM). The method is demonstrated for alloyed Au-Ag NPs of a diameter ranging from 6 to 20 nm produced by laser evaporation of a water-suspended Ag-Au powder mixture of varying composition. Series of EDXS spectra are recorded for 30 NPs from samples with five different Ag:Au ratios revealing Ag depletion from NPs during electron irradiation. By studying the evolution of NPs composition as a function of dose, the initial Ag content for each NP is extrapolated. The rate of Ag depletion is discussed in terms of sputtering and knock-on damage. On average, approximately one Ag atom is lost from the NP for each Ag L X-ray detected. To assess the limitations of microanalysis in these sensitive nanoscale structures, the concept of detectability limit is adapted to our method. This benchmark is then evaluated for Ag in Au-Ag NPs of various sizes and acquisition times. This study should be regarded as a guide for the design of analytical TEM measurements of beam-sensitive NPs.

Nonresonant two-photon mass analyzed threshold ionization and zero kinetic energy photoelectron investigation of the X̃ 2B1 ground state of CH2CO+ and CD2CO+

Rotationally resolved nonresonant two-photon mass analyzed threshold ionization (MATI) and zero kinetic energy (ZEKE) photoelectron spectra of CH2CO+ and CD2CO+ are reported. The spectra are dominated by the origin band and totally symmetric a1 vibrations, ν4 (C=C symmetric stretch) and ν2 (C=O asymmetric stretch) for CH2CO+, and ν1 (C=O asymmetric stretch), ν3 (C=C symmetric stretch), and ν4 (CD2 scissor) for CD2CO+. In addition, several weaker bands are observed in the MATI spectra: ν3 (CH2 scissor) in CH2CO+; b1 vibrations ν5 and ν6 (C=C=O linear bend and CH2 wag) in both isotopomers; b2 vibration ν8 (CD2 rock) for CD2CO+; b2 vibration ν9 (C=C=O linear bend) for CH2CO+; as well as overtones and combination bands. Rotational structure of the origin band is dominated by three very strong ΔKa=±1 bands with 2 orders of magnitude weaker ΔKa=+3 bands. A similar ΔKa=±1 three-band pattern is observed for the a1 vibrational modes. For the b1 modes a single-band pattern resulting from the ΔKa=0 selection rule is...

Membrane order parameters for interdigitated lipid bilayers measured via polarized total-internal-reflection fluorescence microscopy.

Incorporating ethanol in lipid membranes leads to changes in bilayer structure, including the formation of an interdigitated phase. We have used polarized total-internal-reflection fluorescence microscopy (pTIRFM) to measure the order parameter for Texas Red DHPE incorporated in the ethanol-induced interdigitated phase (LβI) formed from ternary lipid mixtures comprising dioleoylphosphatidylcholine, cholesterol and egg sphingomyelin or dipalmitoylphosphatidylcholine. These lipid mixtures have 3 co-existing phases in the presence of ethanol: liquid-ordered, liquid-disordered and LβI. pTIRFM using Texas Red DHPE shows a reversal in fluorescence contrast between the LβI phase and the surrounding disordered phase with changes in the polarization angle. The contrast reversal is due to changes in the orientation of the dye, and provides a rapid method to identify the LβI phase. The measured order parameters for the LβI phase are consistent with a highly ordered membrane environment, similar to a gel phase. An acyl-chain labeled BODIPY-FL-PC was also tested for pTIRFM studies of ethanol-treated bilayers; however, this probe is less useful since the order parameters of the interdigitated phase are consistent with orientations that are close to random, either due to local membrane disorder or to a mixture of extended and looping conformations in which the fluorophore is localized in the polar headgroup region of the bilayer. In summary, we demonstrate that order parameter measurements via pTIRFM using Texas Red-DHPE can rapidly identify the interdigitated phase in supported bilayers. We anticipate that this technique will aid further research in the effects of alcohols and other additives on membranes.