LightBox: A multiwell plate illumination system for photoactive molecule characterization.

Multiwell plates (MWPs) are the workhorses of the life sciences. However, biophotonics research with MWPs is limited, in part due to the lack of equipment suitable for photo-irradiation of photoactive molecules in a MWP-suitable, high-throughput manner, either commercially or through open-source MWP systems. Here we present "LightBox", a calibrated controllable MWP illumination system with broad applications including screening of photoactive molecules and characterization of photocatalytic chemicals. LightBox is a high intensity, accurately controllable, uniform illumination system designed for MWPs with electronics and a control unit that provides a simple and intuitive interface. LightBox can reach intensities of 0.23 mW/mm2 at wavelengths of 405 nm with variance between well sites of <5%. The usefulness of LightBox is demonstrated by assessing the IC50 of a photosensitizing compound using a live/dead assay following simultaneous irradiation of the sample at a range of concentrations, eliminating uncontrolled variables between concentrations and drastically increasing assessment speed.

Effects of maternal anxiety and depression on fetal neuro-development.

BACKGROUND: Fetal development is affected by maternal mental health with research indicating that maternal anxiety and depression are co-morbid; nevertheless differential effects on the fetus have been found. This study examines, prenatally, effects of maternal stress, anxiety and depression on fetal eye-blink reactions to experimental sound and light stimulation. METHODS: Two groups of singleton fetuses (mean 32-weeks gestation) were examined using 4D ultrasound: a control group (N = 14, 7 female) with no stimulation and an experimental group (N = 21, 13 female) exposed to experimental sound, light and cross-modal stimulation. For both groups ultrasound scans were performed and fetal eye-blink was assessed. Mothers completed the Hospital-Anxiety-and-Depression Scale and the Perceived-Stress Scale. Analysis was carried out using Poisson mixed effects modelling. RESULTS: Fetal eye-blink rate during experimental stimulation was significantly and differentially associated with maternal mental health with a 20% increase of fetal eye-blink rate for each unit increase in anxiety score (p = 0.02) and a decrease of 21% of eye blink rate for each unit of increase in depression score (p = 0.02). Sound stimulation but not light stimulation significantly affected blink-rate with fetuses habituating to the stimuli (p < 0.001). LIMITATIONS: Limitations are the relatively small number of fetuses and that a follow up after birth is essential to establish potential long-term effects. CONCLUSIONS: Of clinical importance is the finding that although fetuses are affected by maternal mental health in general here we demonstrate, using eye-blink-rate during stimulation as measure of neuro-development, that fetuses are differentially affected by maternal anxiety and depression with anxiety increasing and depression decreasing fetal reactivity significantly.

Precise spatio-temporal control of rapid optogenetic cell ablation with mem-KillerRed in Zebrafish.

The ability to kill individual or groups of cells in vivo is important for studying cellular processes and their physiological function. Cell-specific genetically encoded photosensitizing proteins, such as KillerRed, permit spatiotemporal optogenetic ablation with low-power laser light. We report dramatically improved resolution and speed of cell targeting in the zebrafish kidney through the use of a selective plane illumination microscope (SPIM). Furthermore, through the novel incorporation of a Bessel beam into the SPIM imaging arm, we were able to improve on targeting speed and precision. The low diffraction of the Bessel beam coupled with the ability to tightly focus it through a high NA lens allowed precise, rapid targeting of subsets of cells at anatomical depth in live, developing zebrafish kidneys. We demonstrate that these specific targeting strategies significantly increase the speed of optoablation as well as fish survival.

Quantitative high dynamic range beam profiling for fluorescence microscopy.

Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.

Micro-endoscope for in vivo widefield high spatial resolution fluorescent imaging.

In this paper we report the design, testing and use of a scannerless probe specifically for minimally invasive imaging of deep tissue in vivo with an epi-fluorescence modality. The probe images a 500 µm diameter field of view through a 710 µm outer diameter probe with a maximum tissue penetration depth of 15 mm specifically configured for eGFP imaging. Example results are given from imaging the pituitary gland of rats and zebrafish hearts with lateral resolution of 2.5 µm.

Investigation of dental samples using a 35MHz focussed ultrasound piezocomposite transducer.

Dental erosion and decay are increasingly prevalent but as yet there is no quantitative monitoring tool. Such a tool would allow earlier diagnosis and treatment and ultimately the prevention of more serious disease and pain. Despite ultrasound having been demonstrated as a method of probing the internal structures of teeth more than 40 years ago, development of a clinical tool has been slow. The aim of the study reported here was to investigate the use of a novel high frequency ultrasound transducer and validate it using a known dental technique. A tooth extracted for clinical reasons was sectioned to provide a sample that contained an enamel and dentine layer such that the enamel-dentine junction (EDJ) was of a varying depth. The sample was then submerged in water and a B-scan recorded using a custom-designed piezocomposite ultrasound transducer with a centre frequency of 35 MHz and a -6 dB bandwidth of 24 MHz. The transducer has an axial resolution of 180 microm and a spatial resolution of 110 microm, a significant advance on previous work using lower frequencies. The depth of the EDJ was measured from the resulting data set and compared to measurements from the sequential grinding and imaging (SGI) method. The B-scan showed that the EDJ was of varying depth. Subsequently, the EDJ measurements were found to have a correlation of 0.89 (p<0.01) against the SGI measurements. The results indicate that high frequency ultrasound is capable of measuring enamel thickness to an accuracy of within 10% of the total enamel thickness, whereas currently there is no clinical tool available to measure enamel thickness.

Individually-addressable flip-chip AlInGaN micropixelated light emitting diode arrays with high continuous and nanosecond output power.

Micropixelated blue (470 nm) and ultraviolet (370 nm) AlInGaN light emitting diode ('micro-LED') arrays have been fabricated in flip-chip format with different pixel diameters (72 microm and 30 microm at, respectively, 100 and 278 pixels/mm(2)). Each micro-LED pixel can be individually-addressed and the devices possess a specially designed n-common contact incorporated to ensure uniform current injection and consequently uniform light emission across the array. The flip-chip micro-LEDs show, per pixel, high continuous output intensity of up to 0.55 microW/microm(2) (55 W/cm(2)) at an injection current density of 10 kA/cm(2) and can sustain continuous injection current densities of up to 12 kA/cm(2) before breakdown. We also demonstrate that nanosecond pulsed output operation of these devices with per pixel onaxis average peak intensity up to 2.9 microW/microm(2) (corresponding to energy of 45pJ per 22ns optical pulse) can be achieved. We investigate the pertinent performance characteristics of these arrays for micro-projection applications, including the prospect of integrated optical pumping of organic semiconductor lasers.

"Aether drag" and moving images.

We contrast the two situations in which either a light beam is incident on a moving medium or a moving optical image is incident on a stationary medium. The principle of relativity suggests that the effects on the light of propagating through the medium should be similar. We find, however, that there are subtle differences which we can understand in terms of the relative alignment of the Poynting and wave vectors. Our analysis and experiments investigate both translational motion and rotation.

Active focus locking in an optically sectioning microscope utilizing a deformable membrane mirror.

A significant challenge for in vivo imaging is to remove movement artifacts. These movements (typically due to either respiration and cardiac-related movement or surface chemical response) are normally limited to the axial direction, and hence features move in and out of the focal plane. This presents a real problem for high-resolution optically sectioned imaging techniques such as confocal and multiphoton microscopy. To overcome this we have developed an actively locked focus-tracking system based around a deformable membrane mirror. This has a significant advantage over more conventional focus-tracking techniques where the microscope objective is dithered, since the active element is not in direct, or indirect, contact with the sample. To examine the operational limits and to demonstrate possible applications for this form of focus locking, sample oscillation and movement are simulated for two different biological applications. We were able to track focus over a 400 microm range (limited by the range of the piezomounted objective) with a rms precision on the focal depth of 0.31 microm +/- 0.05 microm.

Parametric resonance of optically trapped aerosols.

The Brownian dynamics of an optically trapped water droplet are investigated across the transition from over- to underdamped oscillations. The spectrum of position fluctuations evolves from a Lorentzian shape typical of overdamped systems (beads in liquid solvents) to a damped harmonic oscillator spectrum showing a resonance peak. In this later underdamped regime, we excite parametric resonance by periodically modulating the trapping power at twice the resonant frequency. The power spectra of position fluctuations are in excellent agreement with the obtained analytical solutions of a parametrically modulated Langevin equation.

Development of fibre-optic confocal microscopy for detection and diagnosis of dental caries.

We report on the development of a fibre-optics-based confocal imaging system for the detection and potential diagnosis of early dental caries. A novel optical instrument, capable of recording axial profiles through caries lesions using single-mode optical fibres, has been developed. The practical study illustrates that miniature confocal devices based around single-mode optical fibres may provide additional diagnostic information for the general dental practitioner.

Time-correlated single-photon counting fluorescence lifetime confocal imaging of decayed and sound dental structures with a white-light supercontinuum source.

We report the demonstration of time-correlated single-photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white-light supercontinuum excitation source. By using a 100 fs-pulsed Ti:Sapphire laser with a low-frequency chirp to pump a 30-cm long section of photonic crystal fibre, a ps-pulsed white-light supercontinuum was created. Optical bandpass interference filters were then applied to this broad-bandwidth source to select the 488-nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but non-infected material. The white-light generation source provides a flexible method of producing variable-bandwidth visible and ps-pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.

Optical ferris wheel for ultracold atoms.

We propose a versatile optical ring lattice suitable for trapping cold and quantum degenerate atomic samples. We demonstrate the realisation of intensity patterns from pairs of Laguerre-Gauss (exp(i??) modes with different ? indices. These patterns can be rotated by introducing a frequency shift between the modes. We can generate bright ring lattices for trapping atoms in red-detuned light, and dark ring lattices suitable for trapping atoms with minimal heating in the optical vortices of blue-detuned light. The lattice sites can be joined to form a uniform ring trap, making it ideal for studying persistent currents and the Mott insulator transition in a ring geometry.

Adaptive optics for enhanced signal in CARS microscopy.

We report the use of adaptive optics with coherent anti-Stokes Raman scattering (CARS) microscopy for label-free deep tissue imaging based on molecular vibrational spectroscopy. The setup employs a deformable membrane mirror and a random search optimization algorithm to improve signal intensity and image quality at large sample depths. We demonstrate the ability to correct for both system and sample-induced aberrations in test samples as well as in muscle tissue in order to enhance the CARS signal. The combined system and sample-induced aberration correction increased the signal by an average factor of approximately 3x for the test samples at a depth of 700 microm and approximately 6x for muscle tissue at a depth of 260 microm. The enhanced signal and higher penetration depth offered by adaptive optics will augment CARS microscopy as an in vivo and in situ biomedical imaging modality.

Advances in laser sources for confocal and multiphoton microscopy.

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.

Evaluation of enamel dental restoration interface by optical coherence tomography.

Evaluation of molar dental restorations on enamel is performed using optical coherence tomography (OCT) with 10 microm resolution. Images of approximately 50 microm failure gaps in the restorations are demonstrated and the OCT images are compared with x-ray and optical microscopy pictures. The results demonstrate the potential of the technique for clinical evaluation of dental restorations.

Use of confocal and multiphoton microscopy for the evaluation of micro-optical components and emitters.

We report on the application of confocal and multiphoton microscopic techniques for the evaluation of the latest generation of micro optical components. The optical emitting characteristics of arrays of matrix addressable GaN micrometer-sized light emitting diodes (micro-LEDs) have been measured using a commercial confocal microscope utilising the LEDs' own emission along with reflection confocal microscopy to determine the surface structure. Multiphoton induced luminescence from the 10-20-micron diameter emitters has also been used to examine the structure of the device and we compare this with electrically induced emission. In related work, the optical properties of micro lens arrays (10-100-micron diameter) fabricated in SiC, Sapphire, and Diamond have been determined using transmission confocal microscopy. Such optical microscopy techniques offer a simple, non-destructive method to determine the structure and performance of such novel devices.

A review of potential new diagnostic modalities for caries lesions.

This paper aims to present a simple overview of potential new diagnostic methods for dental caries. There are several novel methods of caries detection (with potential application to diagnosis) which have been proposed in the last few years, in addition to those that are gaining some commercial exposure and clinical acceptance. For the most part, these methods have been demonstrated in laboratories and are generally many years away from routine clinical application. They include multi-photon imaging, infrared thermography and infrared fluorescence, optical coherence tomography, ultrasound, and terahertz imaging.

Two-photon microscopy of fura-2-loaded cardiac myocytes with an all-solid-state tunable and visible femtosecond laser source.

We report a robust and reliable platform source for visible-wavelength multiphoton microscopy that is based on nonlinear optical methods. We demonstrate a synchronously pumped, singly resonant optical parametric oscillator with simultaneous intracavity third-order quasi-phase matching in a single crystal that generates continuously tunable, visible, and femtosecond-pulsed radiation. The application of the system is demonstrated by two-photon laser-scanning fluorescence microscopy of rabbit cardiac myocytes loaded with the fluorescent Ca2+ indicator fura-2.