The characterisation of InGaN/GaN quantum well light emitting diodes

By focussing on the properties of InGaN/GaN quantum well (QW) LEDs the key physical processes relevant to all InGaN/GaN light emitters are studied. These include the strength of the piezoelectric field, the important current pathways and the effect doping densities and anneal temperatures have on device performance. Photocurrent absorption spectra, of 35A Ino.1Gao.9N QW LEDs, were measured for a range of reverse bias. A bias of 8.5 V was necessary to counteract the affect of the internal piezoelectric field. Using this value and an appropriate approximation for the depletion width of a p-z'-n junction the calculated piezoelectric field was (1.9 0.15) MVcm"1, in good agreement with 1.8 MVcm"1 calculated using piezoelectric constants interpolated from the binaries. The absorption spectra of 26A wide Ino.i6Gao.84N QW LEDs exhibit a band tail extending to low photon energies whereas emission occurs from the low energy side of this band tail, suggesting emission occurs from localised potential minima. Light-current (LI) characteristics, measured as a function of temperature, are sublinear and exhibit a distinctive temperature dependence. These characteristics are explained in terms of drift leakage which is exacerbated due to the large acceptor activation energy in Mg doped GaN. The data was simulated using a drift diffusion model and good agreement between experimental and simulated results is obtained providing the model includes the band tailing. Emission and absorption spectra and LI characteristics were measured for 25A Ino.1Gao.9N/GaN QW LEDs subject to four different anneal temperatures of 700, 750, 850 and 900°C. Using a drift diffusion model, incorporating different acceptor concentrations to simulate the effect of different anneal temperatures, good agreement was achieved between the trends seen in the experimental results and those produced by the simulations. This confirms the important roles drift leakage and thermal annealing have on these devices

Bessel-beam hyperspectral CARS microscopy with sparse sampling: enabling high-content high-throughput label-free quantitative chemical imaging

Microscopy-based high-content and high-throughput analysis of cellular systems plays a central role in drug discovery. However, for contrast and specificity, the majority of assays require a fluorescent readout which always comes with the risk of alteration of the true biological conditions. In this work, we demonstrate a label-free imaging platform which combines chemically specific hyperspectral coherent anti-Stokes Raman scattering microscopy with sparse sampling and Bessel beam illumination. This enabled us to screen multiwell plates at high speed, while retaining the high-content chemical analysis of hyperspectral imaging. To demonstrate the practical applicability of the method we addressed a critical side effect in drug screens, namely, drug-induced lipid storage within hepatic tissue. We screened 15 combinations of drugs and neutral lipids added to human HepG2 liver cells and developed a high-content quantitative data analysis pipeline which extracted the spectra and spatial distributions of lipid and protein components. We then used their combination to train a support vector machine discriminative algorithm. Classification of the drug responses in terms of phospholipidosis versus steatosis was achieved in a completely label-free assay

Dynamic label-free imaging of lipid droplets and their link to fatty acid and pyruvate oxidation in mouse eggs

Mammalian eggs generate most of their ATP by mitochondrial oxidation of pyruvate from the surrounding medium or from fatty acids that are stored as triacylglycerols within lipid droplets. The balance between pyruvate and fatty acid oxidation in generating ATP is not established. We have combined coherent anti-Stokes Raman scattering (CARS) imaging with deuterium labelling of oleic acid to monitor turnover of fatty acids within lipid droplets of living mouse eggs. We found that loss of labelled oleic acid is promoted by pyruvate removal but minimised when β-oxidation is inhibited. Pyruvate removal also causes a significant dispersion of lipid droplets, while inhibition of β-oxidation causes droplet clustering. Live imaging of luciferase or FAD autofluorescence from mitochondria, suggest that inhibition of β-oxidation in mouse eggs only leads to a transient decrease in ATP because there is compensatory uptake of pyruvate into mitochondria. Inhibition of pyruvate uptake followed by β-oxidation caused a similar and successive decline in ATP. Our data suggest that β-oxidation and pyruvate oxidation contribute almost equally to resting ATP production in resting mouse eggs and that reorganisation of lipid droplets occurs in response to metabolic demand

Quantitative spatiotemporal chemical profiling of individual lipid droplets by hyperspectral CARS microscopy in living human adipose-derived stem cells

There is increasing evidence showing that cytosolic lipid droplets, present in all eucaryotic cells, play a key role in many cellular functions. Yet their composition at the individual droplet level and how it evolves over time in living cells is essentially unknown due to the lack of suitable quantitative non-destructive measurement techniques. In this work we demonstrate the ability of label-free hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy, together with a quantitative image analysis algorithm developed by us, to quantify the lipid type and content in vol:vol concentration units of individual lipid droplets in living human adipose-derived stem cells during differentiation over 9 days in media supplemented with different fatty acids. Specifically, we investigated the addition of the poly-unsaturated linoleic and alpha-linolenic fatty acids into the normal differentiation medium (mostly containing mono-unsaturated fatty acids). We observe a heterogeneous uptake which is droplet-size dependent, time dependent, and lipid dependent. Cells grown in linoleic acid-supplemented medium show the largest distribution of lipid content across different droplets at all times during differentiation. When analyzing the average lipid content, we find that adding linoleic or alpha-linolenic fatty acids at day 0 results in uptake of the new lipid components with an exponential time constant of 22±2hr. Conversely, switching lipids at day 3 results in an exponential time constant of 60±5hr. These are unprecedented findings, exemplifying that the quantitative imaging method demonstrated here could open a radically new way of studying and understanding cytosolic lipid droplets in living cells

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos

Heterodyne dual-polarization epi-detected CARS microscopy for chemical and topographic imaging of interfaces

Coherent Raman Scattering (CRS) has emerged in the last decade as a powerful multiphoton microscopy technique offering chemically-specific label-free imaging in real time with high three-dimensional spatial resolution. Many technical realizations of CRS microscopy have been proposed to remove, suppress, or account for the non-resonant background in the nonlinear susceptibility which complicates spectral analysis and reduces image contrast. Here, we demonstrate coherent anti-Stokes Raman scattering microscopy using a dual-polarization balanced heterodyne detection in epi-geometry (eH-CARS), providing background-free chemically-specific image contrast for nanoparticles and interfaces, shot-noise limited detection, and phase sensitivity. We show the sensitivity and selectivity of eH-CARS in comparison with forward CARS and stimulated Raman scattering (SRS) on polystyrene beads in agarose gel. As an important biologically-relevant application, we demonstrate eH-CARS imaging of individual lipid bilayers with high contrast and topographic sensitivity

Imaging lipids in living mammalian oocytes and early embryos by coherent Raman scattering microscopy

Many promising techniques proposed to monitor gamete developmental potential and quality are invasive and not realistically useful in clinical practise. Hence, there is increasing interest in the development of non-invasive imaging methods that can be applied to mammalian eggs and early embryos. Recent studies have shown that mammalian oocyte and embryo viability are closely associated with their metabolic profile, relying entirely on mitochondria as a source of ATP. Fatty acids, stored in intracellular lipid droplets, are an important source of ATP. We have recently demonstrated the use of Coherent Anti-stokes Raman Scattering (CARS) microscopy to allow chemically-specific, label-free imaging of lipid droplets, with high three-dimensional spatial resolution. Here, we summarize our main findings when using CARS to examine the number, size, and 3D spatial distribution of lipid droplets in mouse eggs and early embryos. Quantitative analysis showed statistically significant differences during oocyte maturation and early embryo development. Notably, CARS imaging did not compromise maturation or development. In mouse oocytes that had been subjected to alterations in mitochondrial metabolism we found that the spatial distribution pattern of lipid droplets was also altered. In addition, differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids were detected using CARS hyperspectral imaging. We also imaged bovine oocytes, and found that lipid droplets appear to be larger and with less spatial aggregation than in mouse oocytes, possibly reflecting the fact that different species metabolise lipids differently. These data suggest that CARS microscopy is a promising non-invasive technique for assessing specific aspects of the metabolic profile of living mammalian eggs and early embryos, which could be potentially linked to their quality and viability

Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti:Sa laser

We have developed a multimodal multiphoton laser-scanning microscope for cell imaging featuring simultaneous acquisition of differential Coherent Antistokes Raman Scattering (D-CARS), two-photon fluorescence (TPF) and second harmonic generation (SHG) using a single 5 fs Ti:Sa broadband (660-970 nm) laser. The spectral and temporal pulse requirements of these modalities were optimized independently by splitting the laser spectrum into three parts: TPF/SHG excitation (> 900 nm), CARS Pump excitation (< 730 nm), and CARS Stokes excitation (730-900 nm). In particular, by applying an equal linear chirp to pump and Stokes pulses using glass dispersion we achieved a CARS spectral resolution of 10 cm(-1), and acquired CARS images over the 1200-3800 cm(-1) vibrational range selected by the time delay between pump and Stokes. A prism pulse compressor in the TPF/SHG excitation was used to achieve Fourier limited 30 fs pulses at the sample for optimum TPF and SHG. D-CARS was implemented with few passive optical elements and enabled simultaneous excitation and detection of two vibrational frequencies with a separation adjustable from 20 cm(-1) to 150 cm(-1) for selective chemical contrast and background suppression. The excitation/detection set-up using beam-scanning was built around a commercial inverted microscope stand providing conventional bright-field, differential interference contrast and epi-fluorescence for user-friendly characterization of biological samples. Examples of CARS hyperspectral images and simultaneous acquisition of D-CARS, TPF and SHG images in both forward and epi-direction are shown on HeLa cells, stem-cell derived human adipocytes and mouse tissues

Heterodyne dual-polarization epi-detected CARS microscopy for chemical and topographic imaging of interfaces

We present a label-free vibrational microscopy technique recently developed by us, which offers backgroundfree chemically-specific image contrast, shot-noise limited detection, and phase sensitivity enabling topographic imaging of interfaces. The technique features interferometric heterodyne detection of coherent anti-Stokes Raman scattering (CARS) in epi-geometry, as well as multi-modal acquisition of stimulated Raman scattering and forward-emitted CARS intensity in the same instrument. As an important biologically-relevant application, epi-detected heterodyne CARS imaging of individual lipid bilayers is demonstrated. We show that we can resolve a single lipid bilayer, distinct from a double bilayer, and measure the phase of its susceptibility, which provides information about the topography of the bilayer with nanometer resolution. As an additional application example, we show imaging of silicon oil droplets surrounded by an aqueous environment at the glass-water interface, where three different signal generation pathways are distinguished. Our epi-detected heterodyne CARS microscope setup thus paves the way to exciting new experiments pushing the sensitivity and resolution limits of vibrational microscopy to the nanoscale

Correlative light electron microscopy using small gold nanoparticles as single probes

Correlative light electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave-mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 um size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods