Stable relativistic/charge-displacement channels in ultrahigh power density (approximately 10(21 W/cm3) plasmas.

Robust stability is a chief characteristic of relativistic/charge-displacement self-channeling. Theoretical analysis of the dynamics of this stability (i) reveals a leading role for the eigenmodes in the development of stable channels, (ii) suggests a technique using a simple longitudinal gradient in the electron density to extend the zone of stability into the high electron density/high power density regime, (iii) indicates that a situation approaching unconditional stability can be achieved, (iv) demonstrates the efficacy of the stable dynamics in trapping severely perturbed beams in single uniform channels, and (v) predicts that approximately 10(4) critical powers can be trapped in a single stable channel. The scaling of the maximum power density with the propagating wavelength lambda is shown to be proportional to lambda-4 for a given propagating power and a fixed ratio of the electron plasma density to the critical plasma density. An estimate of the maximum power density that can be achieved in these channels with a power of approximately 2 TW at a UV (248 nm) wavelength gives a value of approximately 10(21) W/cm3 with a corresponding atomic specific magnitude of approximately 60 W/atom. The characteristic intensity propagating in the channel under these conditions exceeds 10(21) W/cm2.

Measurement of 160-fs, 248-nm pulses by two-photon fluorescence in fused-silica crystals.

Measurements of 160-fs, 248-nm ultrashort pulses are obtained through a two-photon fluorescence measurement based on the two-photon-induced color-center fluorescence in fused-silica crystals. The method proved to be reliable and advantageous in comparison with two-photon fluorescence techniques employing other materials, both solid state and gaseous.

Short-pulse amplification at 745 nm in Ti:sapphire with a continuously tunable regenerative amplifier.

We describe the design of a continuously tunable Ti:sapphire regenerative amplifier that is capable of sustaining amplification over a wavelength range from 730 nm to more than 800 nm. The amplifier cavity is tuned with a prism pair in combination with a spherical mirror. It sustains an oscillation bandwidth of 5 nm and produces a chirped output pulse energy of 1.2 mJ compressible to a duration of 140 fs at 745 nm. We present a calculation of the theoretical bandwidth.

Biomedical three-dimensional holographic microimaging at visible, ultraviolet and X-ray wavelengths.

A new imaging technology is under advanced development that has several key advantages over conventional forms of microimaging performed with standard light microscopes, confocal light microscopes, and electron microscopes. The image created by this microscope possesses several unique features: It is intrinsically three-dimensional; it can be formed with very high contrast, a characteristic of the phase-sensitive nature of the recording; the information contained in the image is obtained in a single exposure of the specimen, a feature that eliminates the accumulation of damage to living systems that can occur with techniques utilizing multiple exposures; the method of image construction is fundamentally free from aberration (distortion), thereby obviating the need to employ complex procedures for correction; the exact focal plane of any optical section is digitally determined through computation and is not based on any mechanical adjustments; and the principles of operation, including the computational processes and modalities of image presentation, are uniform over the full range of spectral coverage spanning from the visible (approximately 500 nm) to the X-ray (approximately 0.3 nm) regions. The application of this new technology is expected to open new cost-effective avenues to the understanding, prevention and treatment of broad areas of human disease.

Photosensitization of aqueous model systems by hypericin.

Absorption and fluorescence measurements of purified hypericin (HY) were made in various media. Photosensitization of two aqueous systems was investigated: resealed red blood cell membranes (ghosts) and hen lysozyme (Lys). Solubilization of HY by ghost membranes was shown by means of diffuse reflectance spectroscopy. Visible light irradiation of the ghosts incorporating HY led to lipid peroxidation with evidence of singlet oxygen involvement. A binding model applicable for insoluble ligands is indicative of strong HY binding to HSA. The HY-HSA complex photosensitized inactivation of Lys. The pseudo-first-order reaction kinetics with protection by azide ion are consistent with a Type II mechanism mediated by singlet oxygen. The results are discussed in the context of the HY photodynamic and antiretroviral activities.

Fourier-transform holographic microscope.

We describe a holographic microscope with a spatial resolution approaching the diffraction limit. The instrument uses a tiny drop of glycerol as a lens to create the spherically diverging reference illumination necessary for Fourier-transform holography. Measurement of the point-spread function, which is obtained by imaging a knife edge in dark-field illumination, indicates a transverse resolution of 1.4 microm with wavelength lambda = 514.5 nm. Longitudinal resolution is obtained from the holograms by the numerical equivalent of optical sectioning. We describe the method of reconstruction and demonstrate the microscope's capability with selected biological specimens. The instrument offers two unique capabilities: (1) it can collect three-dimensional information in a single pulse of light, avoiding specimen damage and bleaching; and (2) it can record three-dimensional motion pictures from a series of light pulses. The conceptual design is applicable to a broad range of wavelengths and we discuss extension to the x-ray regime.

Photophysics of metalloazurins.

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.

Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study.

The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A.

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins.

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.

A new component in protein fluorescence.

Tryptophyl residues in proteins absorb at longer wavelengths than tyrosyl residues, thus the tryptophyl fluorescence can be selectively excited. In addition, tryptophyl residues have a fluorescence maximum at much longer wavelengths than tyrosyl residues and are the predominant source of fluorescence in the long-wavelength region. The contribution of tyrosyl fluorescence to protein fluorescence can be determined by exploiting these spectral properties. The fluorescence of the tyrosyl residues in native human serum albumin is different from the fluorescence of N-acetyl-L-tyrosinamide; the spectral maximum is at a longer wavelength, and the spectral width is greater. This difference is caused by a second component with a maximum at 345 nm. The excitation spectrum of the 345-nm component is similar to that of the normal 304-nm tyrosyl component. The 345-nm component is largely absent from denatured serum albumin. An excited singlet state protolysis from the buried tyrosyl residues explains the appearance of the 345-nm component. A possible acceptor base is an amino group of buried lysyl residue.

Transfer of singlet energy within trypsin.

Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.

Luminescence studies on Bence-Jones proteins and light chains of immunoglobulins and their subunits.

To provide information on the tertiary structure of the antibody molecule we have investigated the luminescent properties of the light polypeptide chain of human immunoglobulins. The fluorescence and phosphorescence yields, spectra, lifetimes, and anisotropies of a large number of homogeneous light chains, i.e., Bence-Jones proteins and light chains derived from myeloma proteins, were measured. No two proteins gave identical tyrosyl or tryptophyl fluorescence spectra in comparative studies on over 75 proteins belonging to the four basic subgroups of kappa chains and of lambda chains. Spectral differences were apparent even among proteins exhibiting more than 85% amino acid sequence identity. The fluorescence yields of tyrosine and tryptophan vaired 10- and 100-fold, respectively; the Stokes' shift of tryptophan ranged from 328 to 365 nm, but that for tyrosine was apparently invariant (305-307nm). Emission as well as excitation spectra showed tyrosyl and tryptophyl redidues interact minimally or not at all. Fluorescence lifetimes of the tyrosyl and tryptophyl contributions were measured spearately, and the apparent natural lifetimes were calculated. Proteins could be grouped in accordance with similarities in fluorescence lifetimes and fluorescence yields; there was no evident relationship between these groupings and the light chain type (kappa or lambda), amino acid sequence, or tryptophan content. Also apparent were individual differences among kappa light chains and among lambda light chains in respect to their tyrosyl and trptophyl phosphorescence spectra and phosphorescence lifetimes. Certain proteins showed an atypical, short-lived tryptophan phosphorescence decay time. Such variance in the luminescent behavior of the tryptophyl residue(s) indicates a conformational interaction between the V and C domains of light chains. Selective proteolytic cleavage of the light chain into VL and CL fragments permitted the comparison to be made of the luminescent properties of the V and C domains with those of the whole protein. The V domain and intact protein have luminescent features in common, whereas the C domain possesses features distinctive from that of the native protein. Data derived from fluorescence anisotropy spectral studies of intact light chains and their VL-related fragments indicate that energy transfer between tryptophyl residues occurs in the C domain. The results of emission spectroscopic measurements performed at 220 and at 77 K indicate that the observed phophorescence of light chains is mainly from a tryptophyl residue contiguous to a disulfide link. The potential for interdomain interaction in light chains is evidenced by the finding that the orientation of the tryptophyl residue(s) in the V domain can influence the tryptophyl-disulfide ling interactions in the C domain; this interaction may account further for the extensive structural diversity of antibody molecules.

Triplet-triplet energy transfer in alpha-trypsin.

Experiments are reported that demonstrate that light absorbed by ionized tyrosinyl sensitizes the phosphorescence of tryptophanyl residues of native alpha-trypsin. The sensitization effect is abolished when alpha-trypsin is unfolded in guanidine hydrochloride. Under the experimental conditions used, the tryptophan phosphorescence could only have been induced by an electron-exchange interaction. These results, therefore, require that there be at least one ionized tyrosinyl-tryptophanyl pair in the native enzyme and that the distance between the two side chains be sufficiently short to permit electron exchange.