Femtosecond laser inscription of Bragg grating waveguides in bulk diamond.

Femtosecond laser writing is applied to form Bragg grating waveguides in the diamond bulk. Type II waveguides are integrated with a single pulse point-by-point periodic laser modification positioned toward the edge of the waveguide core. These photonic devices, operating in the telecommunications band, allow for simultaneous optical waveguiding and narrowband reflection from a fourth-order grating. This fabrication technology opens the way toward advanced 3D photonic networks in diamond for a range of applications.

Aberration correction for direct laser written waveguides in a transverse geometry.

The depth dependent spherical aberration is investigated for ultrafast laser written waveguides fabricated in a transverse writing geometry using the slit beam shaping technique in the low pulse repetition rate regime. The axial elongation of the focus caused by the aberration leads to a distortion of the refractive index change, and waveguides designed as single mode become multimode. We theoretically estimate a depth range over which the aberration effects can be compensated simply by adjusting the incident laser power. If deeper fabrication is required, it is demonstrated experimentally that the aberration can be successfully removed using adaptive optics to fabricate single mode optical waveguides over a depth range > 1 mm.

Adaptive optimisation of a generalised phase contrast beam shaping system.

The generalised phase contrast (GPC) method provides versatile and efficient light shaping for a range of applications. We have implemented a generalised phase contrast system that used two passes on a single spatial light modulator (SLM). Both the pupil phase distribution and the phase contrast filter were generated by the SLM. This provided extra flexibility and control over the parameters of the system including the phase step magnitude, shape, radius and position of the filter. A feedback method for the on-line optimisation of these properties was also developed. Using feedback from images of the generated light field, it was possible to dynamically adjust the phase filter parameters to provide optimum contrast.

Exploring the depth range for three-dimensional laser machining with aberration correction.

The spherical aberration generated when focusing from air into another medium limits the depth at which ultrafast laser machining can be accurately maintained. We investigate how the depth range may be extended using aberration correction via a liquid crystal spatial light modulator (SLM), in both single point and parallel multi-point fabrication in fused silica. At a moderate numerical aperture (NA = 0.5), high fidelity fabrication with a significant level of parallelisation is demonstrated at the working distance of the objective lens, corresponding to a depth in the glass of 2.4 mm. With a higher numerical aperture (NA = 0.75) objective lens, single point fabrication is demonstrated to a depth of 1 mm utilising the full NA, and deeper with reduced NA, while maintaining high repeatability. We present a complementary theoretical model that enables prediction of the effectiveness of SLM based correction for different aberration magnitudes.

Focussing over the edge: adaptive subsurface laser fabrication up to the sample face.

Direct laser writing is widely used for fabrication of subsurface, three dimensional structures in transparent media. However, the accessible volume is limited by distortion of the focussed beam at the sample edge. We determine the aberrated focal intensity distribution for light focused close to the edge of the substrate. Aberrations are modelled by dividing the pupil into two regions, each corresponding to light passing through the top and side facets. Aberration correction is demonstrated experimentally using a liquid crystal spatial light modulator for femtosecond microfabrication in fused silica. This technique allows controlled subsurface fabrication right up to the edge of the substrate. This can benefit a wide range of applications using direct laser writing, including the manufacture of waveguides and photonic crystals.

Adaptive slit beam shaping for direct laser written waveguides.

We demonstrate an improved method for fabricating optical waveguides in bulk materials by means of femtosecond laser writing. We use an LC spatial light modulator (SLM) to shape the beam focus by generating adaptive slit illumination in the pupil of the objective lens. A diffraction grating is applied in a strip across the SLM to simulate a slit, with the first diffracted order mapped onto the pupil plane of the objective lens while the zeroth order is blocked. This technique enables real-time control of the beam-shaping parameters during writing, facilitating the fabrication of more complicated structures than is possible using nonadaptive methods. Waveguides are demonstrated in fused silica with a coupling loss to single-mode fibers in the range of 0.2 to 0.5 dB and propagation loss <0.4 dB/cm.

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy.

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.

Full spectrum filterless fluorescence microscopy.

In conventional microscopes, fluorescence emission is separated from the backscattered illumination using the Stokes shift, whereby the emission occurs at a longer wavelength to the excitation. Such separation is usually achieved through a combination of wavelength filters that divide the spectrum into mutually exclusive excitation and emission bands. It is therefore impossible in these microscopes to access the full excitation/emission spectrum of the specimen in a single image. We report on a microscope that acquired fluorescence images using illumination across the spectral range 450-680 nm; the full emission spectrum was detected simultaneously across the same range. The microscope was also combined with structured illumination optical sectioning to give three-dimensionally resolved images with improved background rejection. Full spectrum fluorescence images of biological specimens are demonstrated. As this system is more versatile than the standard fluorescence microscope, it could be of benefit in many fluorescence imaging applications.

Specimen-induced distortions in light microscopy.

Specimen-induced aberrations affect the imaging properties in optical 3D microscopy, especially when high numerical aperture lenses are used. Studies on aberrations are often properly concerned with the degradation of image quality such as compromised resolution or reduced signal intensity. Apart from these, aberration effects can also introduce geometric image distortions. The effects, discussed here are particularly strong when thick biological specimens are investigated. Using a high numerical aperture interferometer, we measured wavefront aberrations in transmission mode and quantified geometric distortions associated with specimen-induced aberrations. This assessment for a range of biological specimens allows estimation of the accuracy of spatial measurements. The results show that high-resolution spatial measurements can be significantly compromised by specimen-induced aberrations.

Simple optimization procedure for objective lens correction collar setting.

We present a new method for setting a coverglass correction collar on an objective lens. Axial scans across the interface between the specimen volume and the slide are used together with a quantitative function of merit to determine the optimum setting of the correction collar. The method, which simplifies the adjustment for the user and reduces photobleaching, was implemented within the software environment of a scanning microscope system.

Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry.

Wavefront aberrations caused by the refractive index structure of the specimen are known to compromise signal intensity and three-dimensional resolution in confocal and multiphoton microscopy. However, adaptive optics can measure and correct specimen-induced aberrations. For the design of an adaptive optics system, information on the type and amount of the aberration is required. We have previously described an interferometric set-up capable of measuring specimen-induced aberrations and a method for the extraction of the Zernike mode content. In this paper we have modelled specimen-induced aberrations caused by spherical and cylindrical objects using a ray tracing method. The Zernike mode content of the wavefronts was then extracted from the simulated wavefronts and compared with experimental results. Aberrations for a simple model of an oocyte cell consisting of two spherical regions and for a model of a well-characterized optical fibre are calculated. This simple model gave Zernike mode data that are in good agreement with experimental results.

Low-cost, frequency-domain, fluorescence lifetime confocal microscopy.

We describe the theory and implementation of a frequency-domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications-type, radio-frequency electronics are used to provide inexpensive modulation references and to perform phase-sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.

Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids.

Adeno-associated virus (AAV) vectors are highly efficient tools for use in gene therapy. Current production methods rely on plasmid transfection and are not generally considered amenable to scale-up. To improve recombinant AAV (rAAV) vector production in terms of both final titre and simplicity, we constructed recombinant herpes simplex virus (HSV) vectors, either disabled (ICP27 deleted) or nondisabled, encoding the AAV rep and cap genes. We also integrated an rAAVGFP construct into the nondisabled vector and also into a second pair of HSV vectors (disabled and nondisabled) not expressing rep and cap. Transgene incorporation and expression was confirmed by Southern and Western blot, respectively. Optimal double-infection ratios were established for disabled and nondisabled pairs of rep/cap-expressing and rAAVGFP-containing vectors, resulting in up to 1.55 x 10(12) rAAV capsids and 4 x 10(8) expression units from approximately 1 x 10(7) BHK producer cells. Functionality of the prepared vector was confirmed by the detection of abundant green fluorescent protein (GFP) expression following injections of rAAV preparations into the rat brain. This paper therefore describes a simple, efficient, and transfection-free rAAV production process based on the use of HSV and not relying on a proviral cell line that, with appropriate scale-up, could yield quantities of rAAV sufficient for routine clinical use.

Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry.

Confocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.

Temporary remission of disseminated paecilomycosis in a German shepherd dog treated with ketoconazole.

Disseminated mycosis caused by Paecilomyces varioti in a female German shepherd dog presented with chronic forelimb lameness is described. Radiographs of the swollen carpal joint revealed geographic lysis of the radial epiphysis. Diagnosis was based on cytological demonstration of fungal hyphae and chlamydiospores, as well as fungal culture of fluid obtained by arthrocentesis. Temporary remission was characterised by markedly improved clinical signs and laboratory parameters, following treatment with ketoconazole. The dog was euthanased 9 months after the initial diagnosis, following the diagnosis of multifocal discospondylitis. This appears to be the longest described period of temporary remission obtained with treatment in dogs with paecilomycosis. Clinical, clinicopathological and necropsy findings of this disease in another German shepherd dog are briefly described.

Refractive-index-mismatch induced aberrations in single-photon and two-photon microscopy and the use of aberration correction.

We examine the effects of aberrations induced by a refractive index mismatch on the signal level and resolution of single-photon (1-p) and two-photon (2-p), conventional and confocal scanning microscopes. In particular, we consider the aberrations introduced by an interface between oil/glass and water. Resolution is defined in terms of enclosed fluorescence, rather than full-width half-maximum, revealing more useful information for heavily aberrated point spread functions (PSFs). It is shown that, at large focusing depths, the resolution of 2-p conventional and 1-p confocal microscopes are almost identical. The benefits of aberration correction are examined by removing Zernike aberration modes. With aberration correction, the best resolution is found for 1-p confocal and 2-p confocal modes. An approximation based upon geometrical optics is also introduced which shows that the axial resolution of heavily aberrated PSFs is roughly proportional to focusing depth.

Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin.

We consider various strategies for confocal imaging of human skin which seek to reduce the effects of the specimen-induced aberrations. We calculate the spherical aberration introduced by the stratified structure of skin and show how the confocal signal is affected when attempting to image at various depths within the dermis. Using simple methods it is shown how images might be improved by compensating for the induced aberration. The methods include the use of an iris to reduce the pupil area, changing the refractive index of the immersion medium and using a lens with variable coverglass correction.

Closed-loop aberration correction by use of a modal Zernike wave-front sensor.

We describe the practical implementation of a closed-loop adaptive-optics system incorporating a novel modal wave-front sensor. The sensor consists of a static binary-phase computer-generated holographic element, which generates a pattern of spots in a detector plane. Intensity differences between symmetric pairs of these spots give a direct measure of the Zernike mode amplitudes that are present in the input wave front. We use a ferroelectric liquid-crystal spatial light modulator in conjunction with a 4-f system and a spatial filter as a wave-front correction element. We present results showing a rapid increase in Strehl ratio and focal spot quality as the system corrects for deliberately introduced aberrations.

Atypical dermoid sinus in a chow chow dog.

A case of multiple dermoid sinuses in the dorsal cervical and craniothoracic regions in an adult chow chow dog is described. One sinus did not open on the skin surface. This is the first reported case of the condition in this breed and the first time absence of the sinus opening on the skin is described. The use of the term pilonidal sinus to describe this condition is challenged.

Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line.

We report on efficient two-photon fluorescence imaging in beam scanning microscopy by exciting UV dyes at the 647-nm line of a continuous wave ArKr mixed gas laser. For a numerical aperture of 1.4 (oil), we used an illumination power of up to 210 mW at the sample. High-resolution images were obtained for DAPI-labelled cell nuclei within 4-60 s. Our method is a simple two-photon alternative to UV confocal imaging with the potential of becoming a very useful feature of laser scanning microscopy.