We present a four-wave mixing interferometry technique recently developed by us, whereby single non-fluorescing gold nanoparticles are imaged background-free even inside highly heterogeneous cellular environments, owing to their specific nonlinear plasmonic response. The set-up enables correlative four-wave mixing/confocal fluorescence imaging, opening the prospect to study the fate of nanoparticle-biomolecule-fluorophore conjugates and their integrity inside cells. Beyond imaging, the technique features the possibility to track single particles with nanometric position localization precision in 3D from rapid single-point measurements at 1 ms acquisition time, by exploiting the optical vortex field pattern in the focal plane of a high numerical aperture objective lens. These measurements are also uniquely sensitive to the particle in-plane asymmetry and orientation. The localization precision in plane is found to be consistent with the photon shot-noise, while axially it is limited to about 3nm by the nano-positioning sample stage, with an estimated photon shot-noise limit of below 1 nm. As a proof-of- principle, the axial localization is exploited to track single gold nanoparticles of 25nm radius while diffusing across aqueous pockets in a dense agarose gel, mimicking a relevant biological environment.
Coherent anti-Stokes Raman scattering (CARS) microscopy utilises intrinsic vibrational resonances of molecules to drive inelastic scattering of light, and thus eradicates the need for exogenous fluorescent labelling, whilst providing high-resolution three-dimensional images with chemical specificity. Replacement of hydrogen atoms with deuterium presents a labelling strategy that introduces minimal change to compound structure yet is compatible with CARS due to an induced down-shift of the CH2 peak into a region of the Raman spectrum which does not contain contributions from other chemical species, thus giving contrast against other cellular components.
We present our work using deuterated oleic acid to optimise setup of an in-house-developed multimodal, multiphoton, laser-scanning microscope for precise identification of carbon-deuterium-associated peaks within the silent region of the Raman spectrum. Application of the data analysis procedure, factorisation into susceptibilities and concentrations of chemical components (FSC3), enables the identification and quantitative spatial resolution of specific deuterated chemical components within a hyperspectral CARS image. Full hyperspectral CARS datasets were acquired from HeLa cells incubated with either deuterated or non-deuterated oleic acid, and subsequent FSC3 analysis enabled identification of the intracellular location of the exogenously applied deuterated lipid against the chemical background of the cell. Through application of FSC3 analysis, deuterium-labelling may provide a powerful technique for imaging small molecules which are poorly suited to conventional fluorescence techniques.
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.
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.
We demonstrate frequency differential CARS (D-CARS) using femtosecond laser pulses linearly chirped by glass
elements of high group-velocity dispersion. By replicating the Pump-Stokes pair into a pulse train at twice the
laser repetition rate, and controlling the instantaneous frequency difference by glass dispersion, we adjust the
Raman frequency probed by each pair in an intrinsically stable way. The resulting CARS intensities are detected
simultaneously by a single photomultiplier as sum and difference using lock-in detection. We demonstrate
imaging of living cells with strongly suppressed non-resonant background. We also show D-CARS using a single
femtosecond laser source.
We have developed a range of optical biochip devices for conducting live and fixed cell-based assays. The devices
encompass the ability to process an entire assay including fluorescently labelling cells, a microfluidic system to transport
and maintain cells to deliver them to an optical area of the device for measurement, with the possibility of a
incorporating a sorting step in between. On-chip excitation provided by red emitting LED and lasers define the excitation
wavelength of the fluorophore to be incorporated into the assay readout. The challenge for such an integrated
microfluidic optical biochip has been to identify and characterise a longterm fluorescent label suitable for tracking cell
proliferation status in living cells.
Traditional organic fluorophores have inherent disadvantages when considering their use for an on-chip device requiring
longterm cellular tracking. This has led us to utilise inorganic quantum dots (QDots) as fluorophores for on- chip assays.
QDs have unique properties such as photostability, broad absorption and narrow emission spectra and are available in a
range of emission wavelengths including far red. They also have much higher quantum efficiencies than traditional
organic fluorophores thus increasing the possible dynamic range for on-chip detection. Some of the QDots used have the
added advantage of labelling intact cells and being retained and distributed among daughter cells at division, allowing
their detection for up to 6 generations. The use of these QDs off-chip has suggested that they are ideal for live cell, nonperturbing
labelling of division events, whereby over time the QD signal becomes diluted with each generation.
Here we describe the use of quantum dots as live cell tracers for proliferating populations and the potential applications
in drug screening and optical biochip environments.
An optical biochip is being developed for monitoring the sensitivity of biological cells to a range of environmental
changes. Such changes may include external factors such as temperature but can include changes within the suspending
media of the cell. The ability to measure such sensitivity has a broad application base including environmental
monitoring, toxicity evaluation and drug discovery. The device under development, capable of operating with both
suspension and adherent cell populations, employs electrokinetic processes to monitor subtle changes in the physicochemical
properties of cells as environmental parameters are varied. As such, the device is required to maintain cells in
a viable condition for extended periods of time.
The final device will employ integrated optical illumination of cells using red emitting LED or laser devices with light
delivery to measurement regions achieved using integrated micro-optical components. Measurements of electrokinetic
phenomena such as dielectrophoresis and electrorotation will be achieved through integrated optical detectors.
Environmental parameters can be varied while cells are actively retained within a measurement structure. This enables
the properties and sensitivity of a cell population to be temporally tracked.
The optical biochip described here uses a combination of microfabrication techniques including photolithographic and
laser micromachining processes. Here we describe the design and manufacturing processes to create the components of
the environmental monitoring strutures of the optical biochip.
We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence
intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent
species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength
corrugations fabricated on a thin gold film. We have made fluorescence intensity measurements of both an organic dye
(Cy5), and immobilized and fluorescently labeled (with 705 nm emitting quantum dots), mammalian tumor cells in
contact with the active surface. Here we show that the presence of the periodic grating can be used to control both the
excitation and fluorescence generation process itself. We demonstrate that the gratings convert evanescent surface optical
modes into well-defined beams of radiation in the far-field and at the surface of the device this produces highly
contrasting regions of fluorescence excitation providing regions of high spatial selectivity.
We have measured the pulsed light-current characteristics of a series of InGaN/GaN quantum well light-emitting diodes which were annealed post-growth at different temperatures as a function of their operating temperature. The light output at a fixed current density increases with the temperature of measurement, reaches a maximum and then decreases for all the diodes. The measurement temperature at which the maximum light output occurs and the magnitude of the light output depend on the post-growth thermal anneal temperature. The thermal anneal temperature is thought to affect the acceptor concentration in the p-doped cap layer, which also changes the carrier mobility. A simulation, incorporating carrier leakage, is used to reproduce the experimental behavior where the acceptor concentration is changed to represent the effects of the different anneal temperatures.
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