STM of isolated complexes of R-Phycoerythrin: direct observation of the gamma-subunit at the centre of the complex.

Scanning tunnelling microscopy was used to study the topography of single complexes of the photosynthetic protein R-Phycoerythrin and to determine the location of the gamma-subunit. This study has resolved an ambiguity in the positioning of the gamma-subunit from X-ray crystal structure analysis which arose from the symmetry-related averaging of the X-ray diffraction pattern which led to a loss of resolution.

Three-dimensional second-harmonic generation imaging with femtosecond laser pulses.

A three-dimensional reflectance scanning optical microscope based on the nonlinear optical phenomenon of second-harmonic generation is presented. A mode-locked Ti:sapphire laser producing <90-fs pulses at approximately 790 nm was used, and the images were constructed by scanning of an object, which possessed local second-order nonlinearity, relative to a focused spot from the laser. The second-harmonic light at approximately 395 nm generated by the specimen was separated from the fundamental beam by use of dichroic and interference filters and was detected by a photodiode.

Time-resolved spectroscopic fluorescence imaging, transient absorption and vibrational spectroscopy of intact and photo-inhibited photosynthetic tissue.

Fluorescence, absorption and vibrational spectroscopic techniques were used to study spinach at the photosystem II (PS II), chloroplast and cellular levels and to determine the effects and mechanisms of ultraviolet-B (UV-B) photoinhibition of these structures. Two-photon fluorescence spectroscopic imaging of intact chloroplasts shows significant spatial variations in the component fluorescence spectra in the range 640-740 nm, indicating that the type and distribution of chlorophylls vary markedly with position in the chloroplast. The chlorophyll distributions and excitonic behaviour in chloroplasts and whole plant tissue were studied using picosecond time-gated fluorescence imaging, which also showed UV-induced kinetic changes that clearly indicate that UV-B induces both structural and excitonic uncoupling of chlorophylls within the light-harvesting complexes.

Effects of Ca2+ and EGTA on P680*+ reduction kinetics and O2 evolution of Photosystem II.

We report for the first time significant changes in the P680*+ reduction kinetics of Photosystem II (PS II) in which the 17 and 23 kDa extrinsic polypeptides are intact, in the presence of Ca(2+) or ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) which were added to vary the Ca(2+) concentration from 5 microM to 30 mM. The decrease in the extent of normal P680*+ reduction decay with lifetimes of 40-370 ns and a corresponding increase in the extent of kinetics with lifetimes of 20-220 micros was interpreted as being due to electron transfer from Y(Z) to P680*+ being replaced by slow forward conduction and by processes including P680*+/Q(A)(-) recombination. The question of whether changes in P680*+ reduction kinetics were caused by loss of Ca(2+) from PS II or by direct interaction of EGTA with PS II was addressed by lowering the free-Ca(2+) concentration of suspensions of PS II core complexes by serial dilution in the absence of EGTA.

Evidence for spatially-coherent trans-molecular electron tunnelling through two-dimensional arrays of Photosystem II core complexes.

Localised spatially-coherent electron tunnelling through single Photosystem II complexes and into atomically-flat graphite is observed.

Atomic-resolution STM structure of DNA and localization of the retinoic acid binding site.

Single-molecule imaging by scanning tunnelling microscopy (STM) yields the atomic-resolution (0.6A) structure of individual B-type DNA molecules. The strong correlation between these STM structures and those predicted from the known base sequence indicates that sequencing of single DNA molecules using STM may be feasible.

Picosecond time-gated microscopy of UV-damaged plant tissue.

We demonstrate that picosecond time-gated fluorescence microscopy can be used to monitor subtle changes in the kinetics and spatial distribution of perturbations to the molecular and cellular structure of plant tissue caused by ultraviolet radiation. Single-molecule experiments on Photosystem II and chloroplast preparations give picosecond fluorescence decay kinetics that are similar to those obtained previously on bulk samples. For green plant leaves, localized and well-defined cellular structure is seen for normal material whereas relatively diffuse and non-specific features are seen after UV-irradiation indicating significant UV-induced rupture of the cellular structure.

Optimization of second-harmonic generation microscopy.

We describe the principles and characteristics of second-harmonic generation imaging (SHGI) and explore various methods for optimization of the technique. Second-harmonic imaging is optimized for ultrashort laser pulses, high numerical aperture microscope objectives, a highly sensitive non-descanned large area detector, pseudo-phase-matching, and specimens with large second-order non linearity or which exhibit surface plasmon enhanced phenomena. We also compare and contrast the techniques of SHGI and two-photon excited fluorescence imaging.

Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy.

Simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging is demonstrated using a single femtosecond laser and a scanning microscope. This composite nonlinear microscopic technique was applied to imaging DNA and chromosomes, and it was shown that the two different interaction mechanisms provide complementary information on the structure and nonlinear properties of these biological materials, beyond that achievable using either TPF or SHG imaging alone. The use of separate modes of detection, in reflection and transmission respectively, and the simultaneous nature of the acquisition of the two images allows pure TPF and SHG images in precise registration to be obtained.

Spatial distribution of perylenequinones in lichens and extended quinones in quincyte using confocal fluorescence microscopy.

The application of confocal fluorescence microscopy and microspectrofluorimetry to the characterization of the distribution of organic compounds in bulk lichens and mineral structures is demonstrated. Perylenequinones and extended quinones were chosen as both model compounds and as the naturally occurring fluorophores. These molecules occur, respectively, in corticolous microlichens and in a pink-colored mineral called quincyte.

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level.

Isolation and characterisation of oxygen evolving thylakoids from the marine prokaryote Prochloron didemni.

The present study describes the first successful attempt to isolate oxygen evolving thylakoids and thylakoid fragments from the marine prokaryote Prochloron didemni, a member of the recently discovered group of prochlorophytes. Oxygen evolving thylakoid membranes and fragments were isolated from seawater suspended cells of Prochloron didemni by passage of the cells through a Yeda press and subsequent differential centrifugation of the broken material. Three fractions were collected at 1000 x g, 5000 x g, and 3000 x g and identified by light microscopy as cells (and their fragments), thylakoids and membrane fragments, respectively.

Single-molecule electron tunneling spectroscopy of the higher plant light-harvesting complex LHC II.

Electronic spectroscopy of a single biological molecule is demonstrated with approximately 4 A spatial resolution. The light-harvesting complex II (LHC II), in the ground and photo-excited states, was studied using scanning tunneling microscopy and spectroscopy of intact Photosystem II complexes. Analysis of the spectra indicates that the main mechanisms of tunneling between the STM tip and the surface involve delocalized electronic states of the LHC II and local vibronic states associated with C=C, C=O, C-H, N-H, and O-H groups near the LHC II surface.

Single-molecule high-resolution structure and electron conduction of photosystem II from scanning tunneling microscopy and spectroscopy.

Scanning tunneling microscopy (STM) and spectroscopy (STS) were used to obtain the first direct high resolution ( approximately 0.3 nm) images of single isolated Photosystem II (PS II) molecules, and to determine the supramolecular organization of oxygen-evolving PS II core complexes and PS II membrane fragments including the identification, assignment, location and dimensions of the polypeptide units. Our results predict a unique structural model which we then compare with alternative models.

Photochemical reactions of photosystem II in ethylene glycol.

The behavior of photosystem II (PSII) reactions was investigated under conditions of decreasing water content by the addition of increasing concentrations of ethylene glycol (EG). The photosynthetic activities were measured for PSII samples either directly in aqueous solutions of EG or in the standard buffer medium following EG treatment. Several effects on PSII arise upon exposure to EG.

P680(+) reduction in oxygen-evolving Photosystem II core complexes.

The kinetics of P680(+) reduction in oxygen-evolving spinach Photosystem II (PS II) core particles were studied using both repetitive and single-flash 830 nm transient absorption. From measurements on samples in which PS II turnover is blocked, we estimate radical-pair lifetimes of 2 ns and 19 ns. Nanosecond single-flash measurements indicate decay times of 7 ns, 40 ns and 95 ns.

The effects of ultraviolet irradiation on P680(+) reduction in PS II core complexes measured for individual S-states and during repetitive cycling of the oxygen-evolving complex.

Flash-induced absorbance measurements at 830 nm on both nanosecond and microsecond timescales have been used to characterise the effect of ultraviolet light on Photosystem II core particles. A combination of UV-A and UV-B, closely simulating the spectrum of sunlight below 350 nm, was found to have a primary effect on the donor side of P680. Repetitive measurements indicated reductions in the nanosecond components of the absorbance decay with a concomitant appearance and increase in the amplitude of a component with a 10 μs time constant attributed to slow reduction of P680(+) by Tyrz when the function of the oxygen evolving complex is inhibited.

A comparative study of fabric protection against ultraviolet-induced erythema determined by spectrophotometric and human skin measurements.

Historically, a textile's ability to protect against ultraviolet radiation (UVR)-induced erythema has been based on its UVR transmission. However, due to the nonuniformity of the fabric structure of a textile and its resultant nonuniform transmission, the above prediction may not hold. The fabric protection factors (FPF) of 5 metal meshes, to simulate the weave pattern and yarn dimensions of typical fabrics, and 6 textiles with variable construction (woven and knitted), fibre type and dye were determined using a spectrophotometric assay and human skin testing.