Blue-Green and Ultraviolet micro-LEDs in Neural Imaging and Stimulation

1.   Blue-Green InGaN and Ultraviolet AlGaN Multiple Quantum Well LED Device Fabrication

The Blue-Green LED devices were fabricated from standard epitaxially grown p-n junction GaN heterostructure wafers (on sapphire), with an InGaN/GaN active multiple quantum well region. The 340 nm wavelength LEDs were fabricated from AlGaInN quantum-well p-n junction heterostructures, according to a material design typical for these compact solid state light emitters. Mesa-type LEDs were etched using chlorine-based RIE, photolithography and electron beam deposition were used to make metalic contacts and followed by annealing processes with specific conditions according to materials. The completed devices were flip-chipped for light extraction through the polished sapphire backside. Using an epoxy die bonder, electrically conductive epoxy dots were deposited onto a suitably patterned silicon submount. For heat management and mechanical stability, thermally conductive, electrically insulating, epoxy was deposited in the area surrounding the optical apertures

Each chip containing three 3×2 arrays of devices was mounted onto a commercial DIP package for easy connection to the control circuitry and mounting under the microscope.

2.   Blue-Green micro-LEDs for Dynamical Imaging of Neuronal Circuitry

Imaging of biological structures at cellular level by photonic techniques is an advanced science and craft, most commonly applied in the context of fluorescently labeled probes that are excited by lasers or incoherent lamps within an integrated optical microscope-based imaging system. Micro-LEDs and their arrays enable greater flexibility of the illumination arrangements and with the aim towards compact, portable instrumentation.
The LED assembly was placed below the thin fluidic vessel housing the cultured cells (a perfusion chamber), to enable focusing of the photoexcitation onto single cells (body diameter ~20 mm) with bottom illumination. By carefully choosing the properties of focusing optics, we could achieve one-to-one focusing. The fluorescence emission from the VSD labeled cells was collected upwards by a 40× microscope then projected into a high speed CCD imaging camera. In parallel, and for validating the all-optical approach, the electrical activity of the neural cells was also recorded by membrane penetrating microelectrodes, typical of such invasive electrophysiological techniques (whole-cell patch-clamp recordings). A particular cell was chosen, and an LED from the array was brought under the cell of interest. Fluorescence data was then acquired at 1 KHz using 80x80 element CCD imaging camera.

The top trace shows the electrical signal recorded intracellularly (invasively), displaying “action potential” spikes, whereas the bottom trace shows the optically derived signal corresponding to modulation in fluorescence amplitude of the voltage-sensitive dye. The action potential spikes were triggered by a stimulus voltage pulse applied to the cell-embracing electrolytic (saline) solution. The optical signal corresponds to an approximately 4% change in fluorescence intensity for the 100 mV action potential.

We can envision the use of these micro LED arrays with pixel-size matched/imaged photodiode arrays for dynamical optically recording the activity of multiple neural cells, eventually extending to an entire neuronal circuitry on a chip-scale device. By rapid electrically directed scanning of the LED arrays, neurons in their microcircuitry will be illuminated sequentially with fluorescent signals being detected by the corresponding photodiode elements

(a) Microscope image of hippocampal neural cells in culture (differential contrast mode). (b) Two individual elements of the micro-LED array elements located under two neighboring cells. (c) Fluorescent images when both illuminating LEDs are turned ON, and one is ON at a given gated time, respectively. (d) A concept schematic of the chip-scale optical recording system.

3.  UV micro-LEDs for Activation of Electrical Response in Neural Cells by Photochemical Flash Photolysis

The capability of UV LEDs to uncage neurotransmitter and activate proximate individual hippocampal cultured cells was demonstrated.  The entire LED assembly with the corresponding focusing optics was mounted on a 3D stage that allowed for precise positioning of the LED element below the region of interest. Whole-cell recordings were made on a given neuron.

(a) Image of the neuron with corresponding puffer (left) and patch (right) pipettes, (b) Control experiment with the puffer: red trace indicating the duration of puffer pulse, black trace shows the cell’s response, (c) control experiment with the LED; blue trace indicating the duration for which LED is ON, black trace shows the cell’s response, and (d and e) response recorded from two different cells when both the puffer (red) and LED (blue) were ON.

However, with both the puffer and the LED turned ON (ON times indicated by the red and blue insets), we could readily observe neural cell electrical switching, as shown from the recordings from two cells in figure 9(d) and (e). The ability to trigger action potentials in the neuron indicates that the UV LED effectively uncaged the glutamate that was delivered in the immediate vicinity of the neuron, providing the neurotransmitter excitation action.  (It is also clear from figure (d) and (e) that by carefully controlling the picospritzer conditions, it is possible to achieve shorter latencies between beginning of depolarization and the firing of action potential). Experiments such as these hence show graphically the utility of the nitride UV micro-LEDs as efficient and effective sources for flash photolysis experiments simplifying and reducing the cost and size of conventional setups that require expensive and complicated instrumentation.

4.  Combining Multicore Imaging Fiber with Matrix Addressable Blue/Green LED Arrays for Spatiotemporal Photonic Excitation in the research of visual system development

A.   Blue/green LED array fabrication and image fiber coupling. 
In devising the fabrication strategy for the LED microarray, we needed to innovate several process steps to craft a matrix addressable array from otherwise relatively standard blue or green LED wafer level starting material. The prototype LED array device consists of 100 emitter pixel elements within 500 mm2 area, with 28 mm diameter for an individual element in the array and 50 mm center-to-center spacing for neighboring elements.

The nitride LED microarrays were optically connected (butt-coupled, coupling efficiency ~ 4%) to a 3-foot long 30,000 pixel multicore image fiber (FIGH-30-650S from Fujikura Ltd.) of 600 mm image area diameter with 1.9 mm individual pixel diameter and 3.3mm intercore distance to guide light from the LED array.

B.   Photostimulation of retinal ganglion cells for visual system development studies

10x10 matrix addressable LED array coupled to image fiber has been used to deliver patterned visual stimuli directly onto the retina of developing (Nieuwkoop and Faber, developmental stage 48) Xenopus laevis tadpoles. The retinal sensitivity is especially pronounced in this animal in the blue-green region of the spectrum. The response of optic tectal neurons in the tadpole brain was probed electrically via a standard electrophysiological patch clamp microelectrode technique, under different light excitation patterns from the microscale flexible LED-array driven device, in order to map the so-called visual receptive field (i.e. a single neuron’s response to stimulations from different retinal areas) of tectal neurons. We could measure the degree of convergence of retinal inputs into a given tectal cell. This was done at different developmental stages to study the formation of the retinotopic map during development of the visual system. Additionally, we note that we have been able to apply direct optical stimulation of the brain as well (with finite optical absorption by neural cells in the blue), to elicit some neural response. In sum, that these experiments show the utility of the blue (and green) LED microarrays, integrated with the imaging optical fiber, as a flexible and compact optoelectronic tool for all types of photoexcitation schemes where portability and compactness have premium value.