Niall McAlinden

Thermal and optical characterization of micro-LED probes for in vivo optogenetic neural stimulation

Within optogenetics there is a need for compact light sources that are capable of delivering light with excellent spatial, temporal, and spectral resolution to deep brain structures. Here, we demonstrate a custom GaN-based LED probe for such applications and the electrical, optical, and thermal properties are analyzed. The output power density and emission spectrum were found to be suitable for stimulating channelrhodopsin-2, one of the most common light-sensitive proteins currently used in optogenetics. The LED device produced high light intensities, far in excess of those required to stimulate the light-sensitive proteins within the neurons. Thermal performance was also investigated, illustrating that a broad range of operating regimes in pulsed mode are accessible while keeping a minimum increase in temperature for the brain (0.5°C). This type of custom device represents a significant step forward for the optogenetics community, allowing multiple bright excitation sites along the length of a minimally invasive neural probe.

Optogenetic activation of neocortical neurons in vivo with a sapphire-based micro-scale LED probe

Optogenetics has proven to be a revolutionary technology in neuroscience and has advanced continuously over the past decade. However, optical stimulation technologies for in vivo need to be developed to match the advances in genetics and biochemistry that have driven this field. In particular, conventional approaches for in vivo optical illumination have a limitation on the achievable spatio-temporal resolution. Here we utilize a sapphire-based microscale gallium nitride light-emitting diode (μLED) probe to activate neocortical neurons in vivo. The probes were designed to contain independently controllable multiple μLEDs, emitting at 450 nm wavelength with an irradiance of up to 2 W/mm2. Monte-Carlo stimulations predicted that optical stimulation using a μLED can modulate neural activity within a localized region. To validate this prediction, we tested this probe in the mouse neocortex that expressed channelrhodopsin-2 (ChR2) and compared the results with optical stimulation through a fiber at the cortical surface. We confirmed that both approaches reliably induced action potentials in cortical neurons and that the μLED probe evoked strong responses in deep neurons. Due to the possibility to integrate many optical stimulation sites onto a single shank, the μLED probe is thus a promising approach to control neurons locally in vivo.

Depth-specific optogenetic control in vivo with a scalable, high-density μLED neural probe

Controlling neural circuits is a powerful approach to uncover a causal link between neural activity and behaviour. Optogenetics has been widely adopted by the neuroscience community as it offers cell-type-specific perturbation with millisecond precision. However, these studies require light delivery in complex patterns with cellular-scale resolution, while covering a large volume of tissue at depth in vivo. Here we describe a novel high-density silicon-based microscale light-emitting diode (μLED) array, consisting of up to ninety-six 25 μm-diameter μLEDs emitting at a wavelength of 450 nm with a peak irradiance of 400 mW/mm2. A width of 100 μm, tapering to a 1 μm point, and a 40 μm thickness help minimise tissue damage during insertion. Thermal properties permit a set of optogenetic operating regimes, with ~0.5 °C average temperature increase. We demonstrate depth-dependent activation of mouse neocortical neurons in vivo, offering an inexpensive novel tool for the precise manipulation of neural activity.

Accurate position tracking of optically trapped live cells

Optical trapping is a powerful tool in Life Science research and is becoming common place in many microscopy laboratories and facilities. There is a growing need to directly trap the cells of interest rather than introduce beads to the sample that can affect the fundamental biological functions of the sample and impact on the very properties the user wishes to observe and measure. However, instabilities while tracking large inhomogeneous objects, such as cells, can make tracking position, calibrating trap strength and making reliable measurements challenging. These instabilities often manifest themselves as cell roll or re-orientation and can occur as a result of viscous drag forces and thermal convection, as well as spontaneously due to Brownian forces. In this paper we discuss and mathematically model the cause of this roll and present several experimental approaches for tackling these issues, including using a novel beam profile consisting of three closely spaced traps and tracking a trapped object by analysing fluorescence images. The approaches presented here trap T cells which form part of the adaptive immune response system, but in principle can be applied to a wide range of samples where the size and inhomogeneous nature of the trapped object can hinder particle tracking experiments.

Adaptive optics in an optical trapping system for enhanced lateral trap stiffness at depth

In optical trapping systems the trap stiffness, or spring constant, deteriorates dramatically with trap depth due to optical aberrations and system misalignment. This can severely hamper studies that employ optical tweezers to make accurate quantitative measurements. Here, a deformable membrane mirror is used, in conjunction with a random search algorithm, to correct for these aberrations by optimizing on a merit factor that is directly proportional to the trap stiffness. Previous studies have sought to address this issue but none have used a merit factor that is directly proportional to the trap stiffness. We demonstrate that the lateral trap stiffness, measured with and without aberration correction at increasing depths, improves throughout the trapping range of a conventional trap and allows us to extend the maximum depth at which we can trap from 136 to 166 μm. At a depth of 131 μm, trap stiffness improved by factors of 4.37 and 3.31 for the x- and y-axes respectively. The aberration correction resulted in deformable membrane mirror shapes where a single shape could be applied throughout a wide range of trap depths, showing significant improvement, and had the added benefit of making the lateral trapping forces more uniform in x and y.

New evidence for the influence of step morphology on the formation of Au atomic chains on vicinal Si(111) surfaces

Gold atomic chain structures that grow on singular and vicinal Si(111) surfaces have attracted considerable interest as model systems for exploring quasi–one-dimensional metallic behaviour. The structure of the prototypical Si(111)-5×2-Au system remains controversial, however. Reflection anisotropy spectroscopy provides new, independent evidence supporting a recently proposed three-Au-chain structure. For stepped surfaces, the results provide good evidence that a single Au chain forms adjacent to [] steps and a double Au chain forms adjacent to [] steps. In both cases spectral lineshape and coverage data support a three-chain Si(111)-5×2-Au structure forming over the remainder of the terrace. For all the vicinal Si(111) surfaces, including Si(557) and Si(775), it is the step morphology, and not the terrace width, that determines whether single- or double-Au-chain structures are formed in the region of the steps.

An analytic approach to modeling the optical response of anisotropic nanoparticle arrays at surfaces and interfaces

Anisotropic nanoparticle (NP) arrays with useful optical properties, such as localized plasmon resonances (LPRs), can be grown by self-assembly on substrates. However, these systems often have significant dispersion in NP dimensions and distribution, which makes a numerical approach to modeling the LPRs very difficult. An improved analytic approach to this problem is discussed in detail and applied successfully to NP arrays from three systems that differ in NP metal, shape and distribution, and in substrate and capping layer. The materials and anisotropic NP structures that will produce LPRs in desired spectral regions can be determined using this approach.

The viscoelastic properties of the vitreous humor measured using an optically trapped local probe

We present results demonstrating for the first time that an optically trapped bead can be used as a local probe to measure the variation in the viscoelastic properties of the vitreous humor of a rabbit eye. The Brownian motion of the optically trapped bead was monitored on a fast CCD camera on the millisecond timescale. Analysis of the bead trajectory provides local information about the viscoelastic properties of the medium surrounding the particle. Previous, bulk, methods for measuring the viscoelastic properties of the vitreous destroy the sample and allow only a single averaged measurement to be taken per eye. Whereas, with our approach, we were able to observe local behaviour typical of non-Newtonian and gel-like materials, along with the homogenous and in-homogeneous nature of different regions of the dissected vitreous humor. The motivation behind these measurements is to gain a better understanding of the structure of the vitreous humor in order to design effective drug delivery techniques. In particular, we are interested in methods for delivering drug to the retina of the eye in order to treat sight threatening diseases such as age related macular degeneration.

Viability studies of optically trapped T-cells

We present a viability study of optically trapped live T cell hybridomas. T cells form an important part of the adaptive immune response system which is responsible for fighting particular pathogens or diseases. The cells of interest were directly trapped by a laser operating at a wavelength of 1064 nm and their viability measured as a function of time. Cell death was monitored using an inverted fluorescent microscope to observe the uptake by the cell of the fluorescent dye propidium iodide. Studies were undertaken at various laser powers and beam profiles. There is a growing interest in optically trapping immune cells and this is the first study that investigates the viability of a T cell when trapped using a conventional optical trapping system. In such experiments it is crucial that the T cell remains viable and trapping the cell directly means that any artefacts due to a cell-bead interface are removed. Our motivation behind this experiment is to use optical tweezers to gain a greater understanding of the interaction forces between T cells and antigen presenting cells. Measuring these interactions has become important due to recent theories which indicate that the strength of this interaction may underlie the activation of the T-cell and subsequent immune response.

A minimally invasive optical trapping system to understand cellular interactions at onset of an immune response

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated.