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.

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.

In many studies, the value of the experimentally determined internal piezoelectric field has been reported to be significantly smaller than theoretical values. We believe this is due to an inappropriate approximation for the electric field within the depletion region, which is used in the analysis of experimental data, and we propose an alternative method. Using this alternative, we have measured the strength of the internal field of InGaN p-i-n structures, using reverse bias photocurrent absorption spectroscopy and by fitting the bias dependent peak energy using microscopic theory based on the screened Hartree-Fock approximation. The results agree with those using material constants interpolated from binary values. © 2005 American Institute of Physics. ͓DOI: 10.1063/1.1896446͔The internal field in GaN based quantum wells plays an important role in the operation of nitride-based light emitting diodes and lasers, affecting the emission wavelength, 1 the oscillator strength, 2 and the recombination lifetime, 3 hence an accurate value of the internal field is essential in understanding the properties of these devices. The internal field skews and breaks the symmetry of the well, causing spatial separation of the electron and hole wave functions and hence reduces the electron-hole overlap function. Reported values of the internal field 4,5 for the same nominal indium content vary by more than a factor of two, which is far greater than the expected error due to unintended variations in the indium content. Also there are large reported differences between theoretical and experimental results. 6 The majority of approaches to determine the internal field, have relied upon counteracting the quantum-confined Stark effect with an externally applied reverse bias and measuring properties of the quantum well as a function of this applied reverse bias. The reverse bias acts to oppose the internal field reducing the effect of the induced quantum confined Stark effect. At low bias, the well is skewed due to the internal field. At a critical bias, the contributions from the applied bias and the internal field are equal and opposite. In this case, the overlap of electron and hole wave functions and the ground state electron to heavy hole transition energy are maximized.The value of the externally applied bias to achieve flat band ͑"square-up" the quantum well͒ can then be used to obtain the internal field. The net internal field E in the well ͑E = 0 when the well is square͒ is related to the applied bias V using: 4where E int , L w , N, 0 , d d , and d u are the internal field, the quantum well width, the number of quantum wells, the built-in potential and the depletion and intrinsic widths, respectively. The width of the intrinsic region d u is given by the sum of the multiple well and barrier widths. The internal field E int , is the sum of the fields due to the piezoelectric effect and the spontaneous polarization. The first term of Eq. ͑1͒ is the background field written as the total voltage drop divided by the distance, over ...

In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200-2000/cm) and in the CH stretch region (2600-3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods.

Pulsed light-current characteristics of InGaN/GaN quantum well light-emitting diodes have been measured as a function of temperature, with sublinear behavior observed over the whole temperature range, 130-330 K. A distinctive temperature dependence is also noted where the light output, at a fixed current, initially increases with temperature, before reaching a maximum at 250 K and then decreases with subsequent increases in temperature. On the basis of a drift diffusion model, we can explain the sublinear light-current characteristics and the temperature dependence by the influence of the large acceptor ionization energy in Mg-doped GaN together with a triangular density of states function characteristic of localized states. Without the incorporation of localization effects, we are unable to reproduce the temperature dependence whilst maintaining emission at the observed wavelength. This highlights the importance of localization effects on device performance.

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug delivery vehicles, and contrast agents in vivo. In the quest for superior photostability and bio-compatibility, nanodiamonds (NDs) are considered one of the best choices due to their unique structural, chemical, mechanical, and optical properties. So far, mainly fluorescent NDs have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centers with stable optical properties. Here, we show that single non-fluorescing NDs exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp3 vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and ND size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of NDs internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively.

There is increasing evidence showing that cytosolic lipid droplets, present in all eukaryotic 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 nondestructive 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 polyunsaturated linoleic and alpha-linolenic fatty acids into the normal differentiation medium (mostly containing monounsaturated 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 ± 2 h. Conversely, switching lipids at day 3 results in an exponential time constant of 60 ± 5 h. 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.

Journal of BIOPHOTONICSWe have investigated the ability of dual-frequency Coherent Antistokes Raman Scattering (D-CARS) microspectroscopy, based on femtosecond pulses (100 fs or 5 fs) spectrally focussed by glass dispersion, to distinguish the chemical composition of micron-sized lipid droplets consisting of different triglycerides types (polyunsaturated glyceryl trilinolenate, mono-unsaturated glyceryl trioleate and saturated glyceryl tricaprylate and glyceryl tristearate) in a rapid and label-free way. A systematic comparison of Raman spectra with CARS and D-CARS spectra was used to identify D-CARS spectral signatures which distinguish the disordered poly-unsaturated lipids from the more ordered saturated ones both in the CH-stretch vibration region and in the fingerprint region, without the need for lengthy CARS multiplex acquisition and analysis. D-CARS images of the lipid droplets at few selected wavenumbers clearly resolved the lipid composition differences, and exemplify the potential of this technique for label-free chemically selective rapid imaging of cytosolic lipid droplets in living cells.