Microscale mesoarrays created by dip-pen nanolithography for screening of protein-protein interactions

Using microarrays to probe protein-protein interactions is becoming increasingly attractive due to their compatibility with highly sensitive detection techniques, selectivity of interaction, robustness and capacity for examining multiple proteins simultaneously. The major drawback to using this approach is the relatively large volumes and high concentrations necessary. Reducing the protein array spot size should allow for smaller volumes and lower concentrations to be used as well as opening the way for combination with more sensitive detection technologies. Dip-Pen Nanolithography (DPN) is a recently developed technique for structure creation on the nano to microscale with the capacity to create biological architectures. Here we describe the creation of miniaturised microarrays, 'mesoarrays', using DPN with protein spots 400× smaller by area compared to conventional microarrays. The mesoarrays were then used to probe the ERK2-KSR binding event of the Ras/Raf/MEK/ERK signalling pathway at a physical scale below that previously reported. Whilst the overall assay efficiency was determined to be low, the mesoarrays could detect KSR binding to ERK2 repeatedly and with low non-specific binding. This study serves as a first step towards an approach that can be used for analysis of proteins at a concentration level comparable to that found in the cellular environment.

Laser beam self-focusing in the atmosphere

We propose to exploit a self-focusing effect in the atmosphere to assist delivering powerful laser beams from orbit to the ground. We demonstrate through numerical modeling that when the self-focusing length is comparable with the atmosphere height the spot size on the ground can be reduced well below the diffraction limits without beam quality degradation. The density variation suppresses beam filamentation and provides the self-focusing of the beam as a whole. The use of light self-focusing in the atmosphere can greatly relax the requirements for the orbital optics and ground receivers.

Pain response and follow-up of patients undergoing panretinal laser photocoagulation with reduced exposure times

Purpose - We performed a study of laser panretinal photocoagulation in 20 patients with proliferative retinopathy. We compared short exposure, high-energy laser settings with conventional settings, using a 532?nm, frequency doubled, Neodymium–Yag laser and assessed the patients in terms of pain experienced and effectiveness of treatment. Methods - Twenty patients having panretinal photocoagulation for the first time underwent random allocation to treatment of the superior and inferior hemi-retina. Treatment A used ‘conventional’ parameters: exposure time 0.1?s, power sufficient to produce a visible grey-white burns, spot size 300?µm. The other hemi- retina was treated with treatment B using exposure 0.02?s, 300?µm and sufficient power to have similar endpoint. All patients were asked to evaluate severity of pain on a visual analogue scale. (0=no pain, 10=most severe pain). All patients were masked as to the type of treatment and the order of carrying out the treatment on each patient was randomised. Patients underwent fundus photography and were followed up for 6–45 months. Results - Seventeen patients had proliferative diabetic retinopathy, two had ischaemic central retinal vein occlusion and one had ocular ischaemic syndrome. The mean response to treatment A was 5.11, compared to 1.40 treatment B, on the visual analogue scale, which was statistically significant (P=0.001). All patients preferred treatment B. Further treatments, if required, were performed with treatment B parameters and long-term follow-up has shown no evidence of undertreatment. Conclusions - Shortening exposure time of retinal laser is significantly less painful but equally effective as conventional parameters.

OCT evaluation of visible and subthreshold retinal photocoagulation using 532nm laser

Presentation Purpose:We conducted a study to determine if the spectral domain optical coherence tomography (SD-OCT) could be used as a tool to assess effective delivery of threshold and subthreshold laser burns created using 532nm green wavelength laser. Methods:10 patients planned for panretinal photocoagulation (PRP) for proliferative diabetic retinopathy were included in this study. Before initiating the full PRP, a row of moderately white laser burns as used for conventional PRP was created using 532 nm laser set at threshold power for 0.1 second with 300 microns spot size. Further rows of laser burns were created by altering the duration and power settings on the laser device. The area of the retina irradiated with laser was imaged using the Topcon SD-OCT within a few minutes of laser treatment. Results:Laser burns created using threshold power were seen on the OCT scan in all cases as a homogenous diffuse increase in reflectivity extending across the full thickness of retina (Fig 1). Retinal burns created by lowering the duration of laser pulse to 0.01s were barely visible ophthalmoscopically but were clearly detectable on the OCT scan as a localised, well-defined area of increased tissue reflectivity (Fig 2). Conclusions:OCT is a useful to tool to assess the delivery of laser burns created using the 532 nm green laser. Burns of a subthreshold intensity that may not be visible ophthalmoscopically result in retinal changes that are clearly detectable on OCT imaging. Further studies would be needed to assess the clinical effectiveness of subthreshold laser treatment for retinal vascular diseases using the 532 nm green laser.

Optical trapping with superfocused high-M2 laser diode beam

Many applications of high-power laser diodes demand tight focusing. This is often not possible due to the multimode nature of semiconductor laser radiation possessing beam propagation parameter M2 values in double-digits. We propose a method of 'interference' superfocusing of high-M2 diode laser beams with a technique developed for the generation of Bessel beams based on the employment of an axicon fabricated on the tip of a 100 μm diameter optical fiber with highprecision direct laser writing. Using axicons with apex angle 140º and rounded tip area as small as 10 μm diameter, we demonstrate 2-4 μm diameter focused laser 'needle' beams with approximately 20 μm propagation length generated from multimode diode laser with beam propagation parameter M2=18 and emission wavelength of 960 nm. This is a few-fold reduction compared to the minimal focal spot size of 11 μm that could be achieved if focused by an 'ideal' lens of unity numerical aperture. The same technique using a 160º axicon allowed us to demonstrate few-μm-wide laser 'needle' beams with nearly 100 μm propagation length with which to demonstrate optical trapping of 5-6 μm rat blood red cells in a water-heparin solution. Our results indicate the good potential of superfocused diode laser beams for applications relating to optical trapping and manipulation of microscopic objects including living biological objects with aspirations towards subsequent novel lab-on-chip configurations.

Superfocusing of high-M2 semiconductor laser beams:experimental demonstration

The focusing of multimode laser diode beams is probably the most significant problem that hinders the expansion of the high-power semiconductor lasers in many spatially-demanding applications. Generally, the 'quality' of laser beams is characterized by so-called 'beam propagation parameter' M2, which is defined as the ratio of the divergence of the laser beam to that of a diffraction-limited counterpart. Therefore, M2 determines the ratio of the beam focal-spot size to that of the 'ideal' Gaussian beam focused by the same optical system. Typically, M2 takes the value of 20-50 for high-power broad-stripe laser diodes thus making the focal-spot 1-2 orders of magnitude larger than the diffraction limit. The idea of 'superfocusing' for high-M2 beams relies on a technique developed for the generation of Bessel beams from laser diodes using a cone-shaped lens (axicon). With traditional focusing of multimode radiation, different curvatures of the wavefronts of the various constituent modes lead to a shift of their focal points along the optical axis that in turn implies larger focal-spot sizes with correspondingly increased values of M2. In contrast, the generation of a Bessel-type beam with an axicon relies on 'self-interference' of each mode thus eliminating the underlying reason for an increase in the focal-spot size. For an experimental demonstration of the proposed technique, we used a fiber-coupled laser diode with M2 below 20 and an emission wavelength in ~1μm range. Utilization of the axicons with apex angle of 140deg, made by direct laser writing on a fiber tip, enabled the demonstration of an order of magnitude decrease of the focal-spot size compared to that achievable using an 'ideal' lens of unity numerical aperture. © 2014 SPIE.

InP-based active and passive components for communication systems at 2 μm

Progress on advanced active and passive photonic components that are required for high-speed optical communications over hollow-core photonic bandgap fiber at wavelengths around 2 μm is described in this paper. Single-frequency lasers capable of operating at 10 Gb/s and covering a wide spectral range are realized. A comparison is made between waveguide and surface normal photodiodes with the latter showing good sensitivity up to 15 Gb/s. Passive waveguides, 90° optical hybrids, and arrayed waveguide grating with 100-GHz channel spacing are demonstrated on a large spot-size waveguide platform. Finally, a strong electro-optic effect using the quantum confined Stark effect in strain-balanced multiple quantum wells is demonstrated and used in a Mach-Zehnder modulator capable of operating at 10 Gb/s.

The use of spot simulation imagery for forestry mapping and management

SPOT simulation imagery was acquired for a test site in the Forest of Dean in Gloucestershire, U.K. This data was qualitatively and quantitatively evaluated for its potential application in forest resource mapping and management. A variety of techniques are described for enhancing the image with the aim of providing species level discrimination within the forest. Visual interpretation of the imagery was more successful than automated classification. The heterogeneity within the forest classes, and in particular between the forest and urban class, resulted in poor discrimination using traditional `per-pixel' automated methods of classification. Different means of assessing classification accuracy are proposed. Two techniques for measuring textural variation were investigated in an attempt to improve classification accuracy. The first of these, a sequential segmentation method, was found to be beneficial. The second, a parallel segmentation method, resulted in little improvement though this may be related to a combination of resolution in size of the texture extraction area. The effect on classification accuracy of combining the SPOT simulation imagery with other data types is investigated. A grid cell encoding technique was selected as most appropriate for storing digitised topographic (elevation, slope) and ground truth data. Topographic data were shown to improve species-level classification, though with sixteen classes overall accuracies were consistently below 50%. Neither sub-division into age groups or the incorporation of principal components and a band ratio significantly improved classification accuracy. It is concluded that SPOT imagery will not permit species level classification within forested areas as diverse as the Forest of Dean. The imagery will be most useful as part of a multi-stage sampling scheme. The use of texture analysis is highly recommended for extracting maximum information content from the data. Incorporation of the imagery into a GIS will both aid discrimination and provide a useful management tool.