Philip B. Lukins

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.

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.

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. Microsc. Res. Tech. 47:210–214, 1999.

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.

Direct observation of semiconduction and photovoltaic behaviour in single molecules of the Photosystem II reaction centre

Abstract Scanning tunnelling spectroscopy of single intact Photosystem II molecules was used to directly show that the reaction centre behaves as an organic semiconductor and photovoltaic. The main electron conduction mechanisms observed were (a) semiconduction associated with the normal step-wise linear forward electron transfer pathway OEC–Y z –P680–pheo–Q A –Q B , (b) a photovoltaic effect, (c) tunnelling to surface vibronic and delocalized states, and (d) hopping conduction between polypeptide residues. Parameters for these conduction processes were obtained for both dark and illuminated conditions. The photovoltaic behaviour of the reaction centre contrasts with the photoconductive behaviour of the associated light-harvesting complex LHC-II.

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.

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. There is excellent agreement between the STM and X-ray structures, but subtle differences exist due to radial distortions. We show that the interactions of other molecules with DNA, their binding configurations, and the structure of these complexes can be studied at the single-molecule level. The anti-cancer drug retinoic acid (RA) binds selectively to the minor groove of DNA with up to 6 RA molecules per DNA turn and with the plane of the RA molecule approximately parallel to the DNA symmetry axis. Similar studies for other drug molecules will be valuable in the a priori evaluation of the effectiveness of anti-cancer drugs.

Magneto-optical Cotton—Mouton effect of molecular oxygen. A comparison

Abstract Measurements of the magneto-optical Cotton—Mouton effect at 632.8 nm of O 2 ( 3 Σ) over a range of temperature (≈299–464 K) and pressure (up to 613 kPa) are reported and analysed. The study enables a useful comparison to be made of data obtained under different conditions by the two groups that have undertaken systematic measurements of this effect. Agreement between the experimental results and also the derived molecular properties is found to be satisfactory.

Confocal two-photon spectroscopy of red mercuric iodide

One- and two-photon fluorescence of red mercuric iodide single crystal was studied using confocal laser scanning microscopy and spectroscopy at room temperature. There is a clear difference in the fluorescence spectra for one- and two-photon excitation. With two-photon excitation, a fluorescent band is found at the band gap and its central wavelength shifts toward lower energy with increasing depth below the surface, whereas the fluorescent band for one-photon excitation remains invariant with depth. There is an approximately 70 μm thick defect transition layer near the surface and the defect concentration decreases approximately linearly from the surface to the bulk. The band-gap energy at room temperature is determined as 2.12 eV, which is consistent with previous studies.

Characterization of enhanced emission from excimer laser treated ZnO ceramics using one- and two-photon luminescence spectroscopy and microscopy

The effects of excimer laser irradiation on the surface structure and luminescence of sintered ZnO ceramics were investigated. Photoluminescence spectra of three materials (as-prepared ZnO ceramic, the ceramic blackened by high-energy irradiation and the recovered ceramic treated using lower-energy irradiation) were compared in the visible region at room temperature and 10 K. Each material exhibited a green luminescence band but the blackened ZnO ceramics had the strongest emission in the visible region while the recovered ceramics had significantly shorter decay times.