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. The structures of quincyte and of the lichens Cryptothelium rhodotitton and Graphis hematites are described, and the possibilities of energy dissipation and photoprotection mechanisms in these lichens are discussed. This study also illustrates how, for a wide range of specimens, naturally occurring quinone fluorophores in the specimen can be exploited directly to yield chemical and structural information without using fluorescent labelling. These intrinsic quinonoid compounds have molecular fluorescence yields and laser damage thresholds comparable or superior to common microscopy dyes, and can therefore be used to obtain high-contrast 3D fluorescence imaging without the complications introduced by dye labelling.

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. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.

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. Pigment content, oxygen evolution rate and 77 K fluorescence spectra of these fractions were virtually identical. This finding indicates that the membrane fragments obtained are not enriched in photosystem II. The P680+* reduction kinetics of thylakoid membrane fragments were determined by monitoring flash induced absorption changes at 830 nm and analysing the time course of their decay. The multiphasic relaxation kinetics and their modification by NH2OH were found to be similar to those observed in cyanobacteria and plants. These findings provide an independent line of evidence for the idea of a high conservation of the basic structural and functional pattern of the water oxidising complex in all organisms that perform oxygenic photosynthesis.

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. Conduction within the bulk LHC II is then due to ohmic and hopping conduction as well as tunneling between amino acid residues. Light activation of LHC II occurs via a photoconductive rather than a photovoltaic mechanism. There is a dramatic light-induced increase in the electronic density of states indicating a light-induced enhancement of energy and electron delocalization which is important for the efficient and rapid transfer of excitation energy from LHC II to the Photosystem II reaction center.

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. We show that the combination of quasi-constant-height mode STM operation, STS and suitable choice of sample-substrate preparations can be used to enable investigation of the structure and function of single PS II particles under normal thermodynamic and hydration conditions without the requirement and complications of ordered PS II arrays or crystals. STS was also used to characterize single-molecule electron conduction and tunneling mechanisms in PS II including the semiconduction and photoconduction behavior of the reaction center and photoexcitation effects in the light-harvesting complex LHC II.

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. The technique was then used to characterize the distribution of second-order nonlinearity and microstructure of the nonlinear material lithium triborate.

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. Single-flash measurements show that the nanosecond components have amplitudes which vary with S-state. Increasing UV irradiation inhibited the amplitude of these components without changing their S-state dependence. In addition, UV irradiation resulted in a reduction in the total amplitude, with no change in the proportion of the 10 µs contribution.

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. Both the longer 40 ns and 95 ns components relate to the normal S-state controlled Yz → P680(+) electron transfer dynamics. Our analysis indicates the existence of a 7 ns component which provides evidence for an additional process associated with modified interactions involving the water-splitting catalytic site. Corresponding microsecond measurements show decay times of 4 µs and 90 µs with the possibility of a small component with a decay time of 20-40 µs. The precise origin of the 4 µs component remains uncertain but appears to be associated with the water-splitting center or its binding site while the 90 µs component is assigned to P680(+)-QA (-) recombination. An amplitude and kinetic analysis of the flash dependence data gives results that are consistent with the current model of the oxygen-evolving complex.

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. All 5 meshs and 5 of the 6 textiles allowed spectrophotometric prediction of their FPF compared with off-skin (2 mm) human testing. However, on-skin human testing FPF were generally significantly lower than both the off-skin and spectrophotometric estimates. Although evidence is presented that the nonuniform nature of a textile's structure does influence its FPF predictability, in practice, properly conducted spectrophotometric analysis may yield the most typical indication of the protectiveness of a fabric against UVR-induced erythema.