GaN micro-light-emitting diode arrays with monolithically integrated sapphire microlenses

A microdisk light emitting diode (micro-LED) with a monolithically integrated microlens array was demonstrated. The capability of the lenses in concentrating light emitted from microdisk LEDs was also demonstrated. The focal lengths of the microlenses were determined to be around 44 νm. The emission pattern of the LED emitters was found to be altered by the optical properties of the microlenses. The light emitted by the hybrid device was also found to be less divergent than a broad-area device.published_or_final_versio

Multi-photon attenuation-compensated light-sheet fluorescence microscopy

We thank the UK Engineering and Physical Sciences Research Council for funding (grants EP/P030017/1 and EP/R004854/1), the European Union’s Horizon 2020 Framework Programme (H2020) (675512, BE-OPTICAL), the Danish Council for Independent Research (DFF FTP grant 7017-00021), and the Otto Mønsted Foundation (grant 19-70-0109).Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-of-view in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging. Here we show this promising approach may be implemented in multi-photon (two-photon) light-sheet fluorescence microscopy and, furthermore, can be achieved in a facile manner utilizing a graded neutral density filter, circumventing the need for complex beam shaping apparatus. A “gold standard” system utilizing a spatial light modulator for beam shaping is used to benchmark our implementation. The approach will open up enhanced depth penetration in light-sheet imaging to a wide range of end users.Publisher PDFPeer reviewe

Tandem fluorescence and Raman (fluoRaman) characterisation of a novel photosensitiser in colorectal cancer cell line SW480

The development of new imaging tools, molecules and modalities is crucial to understanding biological processes and the localised cellular impact of bioactive compounds. A small molecule photosensitiser, DC473, has been designed to be both highly fluorescent and to exhibit a strong Raman signal in the cell-silent region of the Raman spectrum due to a diphenylacetylene structure. DC473 has been utilised to perform a range of novel tandem fluorescence and Raman (fluoRaman) imaging probe experiments, enabling a thorough examination of the compound’s cellular localisation, exemplified in colorectal cancer cells (SW480). This multifunctional fluoRaman imaging modality has revealed the presence of the compound in lipid droplets and only weak signal in the cytosol, where both Raman and fluorescence results show the presence of the fluoRaman imaging probe. In addition, Raman microscopy detected the compound in a cell compartment we labelled as the nucleolus, where fluorescence microscopy did not detect the fluoRaman probe due to solvatochromatic effects in a local polar environment. This last finding was only possible with the use of tandem confocal Raman and fluorescence methods. By following the approach detailed herein, incorporation of strong Raman functional groups into fluorophores can enable a plethora of fluoRaman experiments, shedding further light on an imaging probe or potential drug compound’s cellular behaviour and biological activity

Relationship between the morphology of the foveal avascular zone, retinal structure, and macular circulation in patients with diabetes mellitus

Diabetic Retinopathy (DR) is an extremely severe and common degenerative disease. The purpose of this study was to quantify the relationship between various parameters including the Foveal Avascular Zone (FAZ) morphology, retinal layer thickness, and retinal hemodynamic properties in healthy controls and patients with diabetes mellitus (DM) with and with no mild DR (MDR) using Spectral-Domain Optical Coherence Tomography (Spectralis SDOCT, Heidelberg Engineering GmbH, Germany) and the Retinal Function Imager (Optical Imaging, Ltd., Rehovot, Israel). Our results showed a higher FAZ area and diameter in MDR patients. Blood flow analysis also showed that there is a significantly smaller venous blood flow velocity in MDR patients. Also, a significant difference in roundness was observed between DM and MDR groups supporting the development of asymmetrical FAZ expansion with worsening DR. Our results suggest a potential anisotropy in the mechanical properties of the diabetic retina with no retinopathy that may trigger the FAZ elongation in a preferred direction resulting in either thinning or thickening of intraretinal layers in the inner and outer segments of the retina as a result of autoregulation. A detailed understanding of these relationships may facilitate earlier detection of DR, allowing for preservation of vision and better clinical outcomes

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 m range (limited by the range of the piezomounted objective) with a rms precision on the focal depth of 0.31 m±0.05 m

Evaluation of fitness parameters used in an iterative approach to aberration correction in optical sectioning microscopy

A major problem when imaging at depth within a biological sample in confocal or nonlinear microscopy is the introduction of sample induced aberrations. Adaptive optical systems can provide a technique to compensate for these sample aberrations and often iterative optimizations are used to improve on a particular parameter of the image (known as the fitness parameter). In this investigation, using a deformable membrane mirror as the adaptive optic element, we examine the effectiveness of a number of fitness parameters, when used with a genetic algorithm, at determining the optimal mirror shape required to compensate for sample induced aberrations. These fitness parameters are compared in terms of the number of mirror changes required to achieve optimization and the final axial resolution of the optical system. The effect that optimizing each fitness parameter has on the lateral and axial point-spread function is also examined

Adaptive optical methods for in vivo imaging in developing Zebra fish

The humble Zebra fish is rapidly establishing itself as the model of choice for a wide range of biological investigations, in particular at the developing embryo stage. Single Plane Illumination Microscopy (SPIM) has already been shown to be a very powerful method with which to image such samples over extended periods of time. However, the sample is only around 2mm long and its physical structure heavily influenced by the beating heart with the circulation of blood causing movement throughout the fish on the scale of several microns. The paper reports on the development of an active synchronization method, which optically “freezes” motion and significantly reduces light induced toxicity and bleaching within the sample. This has been combined with adaptive optics to remove both instrument and sample induced aberrations enabling live high resolution imaging within the beating heart with micron resolution over extended time periods