High-resolution light-scattering imaging with two-dimensional hexagonal illumination patterns: system implementation and image reconstruction formulations.

Structured illumination microscopy (SIM) was recently adapted to coherent imaging, named structured oblique-illumination microscopy (SOIM), to improve the contrast and resolution of a light-scattering image. Herein, we present high-resolution laterally isotropic SOIM imaging with 2D hexagonal illuminations. The SOIM is implemented in a SIM fluorescence system based on a spatial-light modulator (SLM). We design an SLM pattern to generate diffraction beams at 0° and ± 60.3° simultaneously to form a 2D hexagonal illumination, and undertake calculations to obtain optimal SLM shifts at 19 phases to yield a reconstructed image correctly. Beams of linear and circular polarizations are used to show the effect of polarization on the resolution improvement. We derive the distributions of the electric field of the resultant hexagonal patterns and work out the formulations of the corresponding coherent-scattering imaging for image reconstruction. The reconstructed images of gold nanoparticles (100 nm) confirm the two-fold improvement of resolution and reveal the effect of polarization on resolving adjacent nanoparticles. To demonstrate biological applications, we present the cellular structures of a label-free fixed HeLa cell with improved contrast and resolution. This work enables one to perform high-resolution dual-mode - fluorescence and light-scattering - imaging in a system, and is expected to broaden the applications of SOIM.

Three-beam interference with circular polarization for structured illumination microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) is a wide-field technique that can provide doubled resolution and improved image contrast. In this work, we demonstrate a simple approach to 3D-SIM - using three-beam interference with circular polarization to generate the pattern of structured illumination, so that the modulation contrast is routinely maintained at all orientations without a complicated polarization rotator or mechanical motion. We derive the resultant intensity distribution of the interference pattern to confirm the modulation contrast independent of orientation, and compare the result with those using interfering beams of linear polarization. To evaluate the influence of the modulation contrast on imaging, we compare the simulated SIM images of 100-nm beads. Experimental results are presented to confirm the simulations. Our approach requires merely a λ/4-wave plate to alter the interfering beams from linear to circular polarization. This simplicity together with the use of a spatial light modulator to control the interference pattern facilitates the implementation of a 3D-SIM system and should broaden its application.

WITHDRAWN: Cellular uptake and phototoxicity of bioconjugated fluorescent nanodiamonds.

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Subdiffraction scattered light imaging of gold nanoparticles using structured illumination.

A reflective light-scattering (RLS) microscope with structured illumination (SI) provides subdiffraction resolution and improves the image quality of gold nanoparticles in biological systems. The three-dimensional (3D)-structured pattern is rapidly and precisely controlled with a spatial light modulator and scrambled at the conjugate image plane to increase spatial incoherence. The reconstructed SI-RLS image of 100 nm gold nanoparticles reveals lateral and axial resolutions of approximately 117 and 428 nm. We present a high-resolution image of gold nanoparticles inside a HeLa cell, with improved contrast.

Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy.

Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNFα) with TNFα antibody (anti-TNFα) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 ± 4.9 µm(2) s(-1) and 48.96 ± 2.52 µm(2) s(-1) for Alexa488-TNFα and Atto647N-anti-TNFα were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNFα and Atto647N-anti-TNFα were approximately 4.89 ± 0.24 nm and 9.99 ± 0.52 nm, respectively, which agrees with the values of 5.20 ± 1.23 nm and 9.28 ± 0.86 nm for the native TNFα and the anti-TNFα as determined from dynamic light scattering measurements. For the binding kinetics, association (k(on)) and dissociation (k(off)) rate constants were (1.13 ± 0.08) × 10(4) M(-1) s(-1) and (1.53 ± 0.19) × 10(-3) s(-1) while the corresponding dissociation constant (K(d)) at 25 °C was (1.36 ± 0.10) × 10(-7) M. We believe this is the first report on the binding kinetics for TNFα-antibody recognition in the homogeneous phase. Using this technology, we have shown that controlled experiments can be performed to gain insight into molecular mechanisms involved in the immune response.

Design and construction of a compact end-station at NSRRC for circular-dichroism spectra in the vacuum-ultraviolet region.

A synchrotron-radiation-based circular-dichroism end-station has been implemented at beamline BL04B at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan for biological research. The design and performance of this compact end-station for measuring circular-dichroism spectra in the vacuum-ultraviolet region are described. The linearly polarized light from the beamline is converted to modulated circularly polarized light with a LiF photoelastic modulator to provide a usable wavelength region of 130-330 nm. The light spot at the sample position is 5 mm × 5 mm at a slit width of 300 µm and provides a flux greater than 1 × 10(11) photons s(-1) (0.1% bandwidth)(-1). A vacuum-compatible cell made of two CaF(2) windows has a variable path length from 1.3 µm to 1 mm and a temperature range of 253-363 K. Measured CD spectra of (1S)-(+)-10-camphorsulfonic acid and proteins demonstrated the ability of this system to extend the wavelength down to 172 nm in aqueous solution and 153 nm in hexafluoro-2-propanol.

Photodissociation dynamics of tryptophan and the implication of asymmetric photolysis.

Photodissociation of amino acid tryptophan in a molecular beam at wavelengths of 212.8 and 193 nm, corresponding to excitation to the second and third absorption bands, was investigated using multimass ion imaging techniques. The respective wavelengths also represent excitation to the edge of a positive circular dichroism band and the center of a negative circular dichroism band of L-tryptophan. Only one dissociation channel was observed at both photolysis wavelengths: C(8)NH(6)CH(2)CHNH(2)COOH-->C(8)NH(6)CH(2)+CHNH(2)COOH. Dissociation rates were found to be 1.3x10(6) and 5x10(6) s(-1) at the respective wavelengths. Comparison to theoretical calculation indicates that dissociation occurs on the ground state after internal conversion. Implication of asymmetric photolysis is discussed.

Isotropic image in structured illumination microscopy patterned with a spatial light modulator.

We developed a structured illumination microscopy (SIM) system that uses a spatial light modulator (SLM) to generate interference illumination patterns at four orientations - 0 degrees, 45 degrees, 90 degrees, and 135 degrees, to reconstruct a high-resolution image. The use of a SLM for pattern alterations is rapid and precise, without mechanical calibration; moreover, our design of SLM patterns allows generating the four illumination patterns of high contrast and nearly equivalent periods to achieve a near isotropic enhancement in lateral resolution. We compare the conventional image of 100-nm beads with those reconstructed from two (0 degrees +90 degrees or 45 degrees +135 degrees) and four (0 degrees +45 degrees +90 degrees +135 degrees) pattern orientations to show the differences in resolution and image, with the support of simulations. The reconstructed images of 200-nm beads at various depths and fine structures of actin filaments near the edge of a HeLa cell are presented to demonstrate the intensity distributions in the axial direction and the prospective application to biological systems.

Fluorescence excitation spectra of the b (1)Pi(u), b(') (1)Sigma(u) (+), c(n) (1)Pi(u), and c(n) (') (1)Sigma(u) (+) states of N(2) in the 80-100 nm region.

Fluorescence excitation spectra produced through photoexcitation of N(2) using synchrotron radiation in the spectral region between 80 and 100 nm have been studied. Two broadband detectors were employed to simultaneously monitor fluorescence in the 115-320 nm and 300-700 nm regions, respectively. The peaks in the vacuum ultraviolet fluorescence excitation spectra are found to correspond to excitation of absorption transitions from the ground electronic state to the b (1)Pi(u), b(') (1)Sigma(u) (+), c(n) (1)Pi(u) (with n=4-8), c(n) (') (1)Sigma(u) (+) (with n=5-9), and c(4) (')(v('))(1)Sigma(u) (+) (with v(')=0-8) states of N(2). The relative fluorescence production cross sections for the observed peaks are determined. No fluorescence has been produced through excitation of the most dominating absorption features of the b-X transition except for the (1,0), (5,0), (6,0), and (7,0) bands, in excellent agreement with recent lifetime measurements and theoretical calculations. Fluorescence peaks, which correlate with the long vibrational progressions of the c(4) (') (1)Sigma(u) (+) (with v(')=0-8) and the b(') (1)Sigma(u) (+) (with v(') up to 19), have been observed. The present results provide important information for further unraveling of complicated and intriguing interactions among the excited electronic states of N(2). Furthermore, solar photon excitation of N(2) leading to the production of c(4) (')(0) may provide useful data required for evaluating and analyzing dayglow models relevant to the interpretation of c(4) (')(0) in the atmospheres of Earth, Jupiter, Saturn, Titan, and Triton.

Dissociation of energy-selected c-C2H4S+ in a region 10.6-11.8 eV: threshold photoelectron-photoion coincidence experiments and quantum-chemical calculations.

Dissociation of energy-selected c-C2H4S+ was investigated in a region of 10.6-11.8 eV with a threshold photoelectron-photoion coincidence technique and a synchrotron as a source of vacuum ultraviolet radiation. Branching ratios and average releases of kinetic energy in channels of formation of c-C2H4S+, CH3CS+, and HCS+ were obtained from well-resolved time-of-flight peaks in coincidence mass spectra. Measured average releases of kinetic energy for channel CH3CS+ + H of least energy are substantial and much greater than calculated with quasiequilibrium theory; in contrast, small releases of kinetic energy near the appearance onset for channel HCS+ + CH3 agree satisfactorily with statistical calculations. Calculations of molecular electronic structures and energetics of c-C2H4S+ and C2H3S+ isomers and various fragments and transition states were also performed with Gaussian 3 method to establish dissociation mechanisms. A predicted dissociation energy of 11.05 eV for c-C2H4S --> HCS+ + CH3 agrees with a linearly extrapolated threshold at 10.99+/-0.04 eV and a predicted dissociation mechanism that c-C2H4S+ isomerizes to CH3CHS+ before dissociating to HCS+ + CH3 supports the experimental results. The large releases of kinetic energy for channel CH3CS+ + H might result from a dissociation mechanism according to which c-C2H4S+ isomerizes to a local minimum CH3CSH+ and then dissociates through a transition state to form CH3CS+ + H.

Experiments and quantum-chemical calculations on Rydberg states of H2CS in the region 5.6-9.5 eV.

Absorption spectrum of H(2)CS in the region 5.6-9.5 eV was recorded with a continuously tunable light source of synchrotron radiation. After we subtracted absorption bands of CS(2), our spectrum clearly shows vibrational progressions associated with transitions (1)A(1)(pi,pi*)-X (1)A(1) and (1)B(2)(n,4s)-X (1)A(1) in the region 5.6-6.7 eV. A spectrum from which absorption of C(2)H(4) and CS(2) are subtracted shows several discrete bands in the region 6.9-9.5 eV. A Rydberg state (1)B(2)(n,4p(z)) lying below Rydberg state (1)A(1)(n,4p(y)) is confirmed, and the C-H symmetric stretching (nu(1)) and CH out-of-plane bending (nu(4)) modes for a transition (1)B(2)(n,4s)-X (1)A(1) are identified. New transitions to Rydberg states associated with excitation to 5s-11s, 5p(z)-7p(z), 5p(y)-7p(y), and 3d-6d are identified based on quantum defects and comparison with vertical excitation energies predicted with time-dependent density-functional theory (TD-DFT) and outer-valence Green's-function (OVGF) methods. For lower excited states predictions from these TD-DFT6-31+G calculations agree satisfactorily with experimental values, but for higher Rydberg states the OVGF method using aug-cc-pVTZ basis set augmented with extra diffuse functions yields more accurate predictions of excitation energies.

Experimental and quantum-chemical studies on photoionization and dissociative photoionization of CH2Br2.

The dissociative photoionization of CH2Br2 in a region approximately 10-24 eV was investigated with photoionization mass spectroscopy using a synchrotron radiation source. An adiabatic ionization energy of 10.25 eV determined for CH2Br2 agrees satisfactorily with predictions of 10.26 and 10.25 eV with G2 and G3 methods, respectively. Observed major fragment ions CH2Br+, CHBr+, and CBr+ show appearance energies at 11.22, 12.59, and 15.42 eV, respectively; minor fragment ions CHBr2+, Br+, and CH2+ appear at 12.64, 15.31, and 16.80 eV, respectively. Energies for formation of observed fragment ions and their neutral counterparts upon ionization of CH2Br2 are computed with G2 and G3 methods. Dissociative photoionization channels associated with six observed fragment ions are proposed based on comparison of determined appearance energies and predicted energies. An upper limit of DeltaH0f,298(CHBr+) < or = 300.7 +/- 1.5 kcal mol(-1) is derived experimentally; the adiabatic ionization energy of CHBr is thus derived to be < or = 9.17 +/- 0.23 eV. Literature values for DeltaH0f,298(CBr+) = 362.5 kcal mol(-1) and ionization energy of 10.43 eV for CBr are revised to be less than 332 kcal mol(-1) and 9.11 eV, respectively. Also based on a new experimental ionization energy, DeltaH0f,298(CH2Br2+) is revised to be 236.4 +/- 1.5 kcal mol(-1).