IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005
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Compact Semiconductor Light-Emitting Diodes for
Dynamic Imaging of Neuronal Circuitry
Sowmya Venkataramani, Kristina M. Davitt, Jiayi Zhang, Heng Xu, Yoon-Kyu Song, Barry W. Connors,
and Arto V. Nurmikko, Fellow, IEEE
Abstract—We describe the use of compact green light-emitting
diodes (LEDs) for fluorescence optical imaging and demonstrate
this for dynamic recording from cultured neurons. The efficient
gallium-nitride-based LEDs have individual element sizes comparable to typical biological cells and are operated in proximity
illumination mode for individual neurons. As periodic arrays, and
with direct electrical control of each LED, a spatially periodic
multicellular target can be induced to fluoresce in a predesigned
spatiotemporal sequence, thereby providing a new approach to
dynamic recording of small neural circuits.
Index Terms—Light-emitting diodes (LEDs), optical imaging, patterned hippocampal neurons, voltage-sensitive dye (VSD)
imaging.
I. INTRODUCTION
CCESS to detailed dynamic information of neural circuits is of basic interest to fundamental neuroscience,
as well as to efforts that aim to use such insight to create
new functional computational approaches for man-made devices. A large body of literature exists where direct electrical
access in vitro, up to a few neural cells, is gained by means
of the well-established patch-clamp method of micrometer-size
micropipettes [1], [2]. However, with increasing cell population, the microelectrode methods become cumbersome. Complementing these approaches is an optical recording method
where a potentiometric fluorescent probe (molecular dye) detects the change in membrane potential and reports it as a change
in its intensity or spectrum. Such probes, which are capable of
monitoring the fast electrical signals, have been employed in
numerous studies of cell physiology ever since their introduction in early 1970s [3], [4]. The mechanisms underlying the dye
response involve potential-dependent intramolecular rearrangements or small movements of the dye between the extracellular
and intracellular compartments [5], [6]. The optical approach
A
Manuscript received December 20, 2004 and December 21, 2004; revised
October 26, 2005. This work was supported in part by the National Science
Foundation under Grant BES-0423566 and DARPA.
S. Venkataramani, J. Zhang, and H. Xu are with the Department of
Physics, Brown University, Providence, RI 02912 USA (e-mail: venkataramani@physics.brown.edu; Jiayi Zhang@brown.edu; Heng Xu@brown.edu).
K. M. Davitt and Y.-K. Song are with the Division of Engineering, Brown
University, Providence, RI 02912 USA (e-mail: Kristina Davitt@brown.edu;
yoon-kyu song@brown.edu).
B. W. Connors is with the Department of Neuroscience, Brown University,
Providence, RI 02912 USA (e-mail: Barry Connors@brown.edu).
A. V. Nurmikko is with the Department of Physics and Division
of Engineering, Brown University, Providence, RI 02912 USA (e-mail:
Arto Nurmikko@brown.edu).
Digital Object Identifier 10.1109/JSTQE.2005.857722
affords in principle an avenue to detailed quantitative measurements to elucidate the complex routing of information already
exhibited by just a few interlinked neural cells. The fluorescence
technique usually depends on excitation sources such as bulky
incoherent lamps or large frame lasers, with the optical flux directed at the sample through the objective system of an imaging
microscope [7], [8].
In this paper, we introduce the use of custom-designed, compact light-emitting diodes (LEDs), based on gallium nitride
semiconductors, as a flexible means to perform dynamic optical
imaging of neurons, and demonstrate the technical approach to
cultured hippocampal and cortical cells. In particular, we have
designed and fabricated small area (<100-µm diameter) planar
blue-green LEDs where an individual LED can be aligned to
illuminate a specific fluorescently labeled neural cell in close
physical proximity. We have also fabricated planar LED arrays where independent electrical control of each LED with
microsecond precision is achieved. Illumination of target neurons in a predetermined time sequence can thus enable a new
approach to dynamic recording of small patterned neural circuits (or other spatially organized biological objects equipped
with fluorescent labels). For neural cells that have been cultured
on patterned periodic templates, this approach is part of our
longer-term goal to develop a new type of dynamic imaging approach to neural networks, as well as to achieve an active “chip
scale” interface between a neural and a man-made (optoelectronic) circuit. Here, we show the first results of our research,
with emphasis on the utility of the LEDs as targeted single-cell
excitation sources.
This paper is organized as follows. In Section II, we describe
the design and fabrication of the LED devices and their arrays. Section III describes the optical engineering strategies that
have been used in the initial demonstrations of the new imaging
concept. In Section IV, we describe our approach to culturing neurons, including those on patterned planar templates with
the future aim of creating a controlled two-dimensional neural
architecture. Section V shows examples of the results where
neural dynamics are acquired using the compact LED-based illumination system. As the first step, we have used the LEDs
to record optical signals both from a single hippocampal neuron, as well as pairs of such adjacent cells in culture. We have
achieved a one-to-one focus onto the cells, thereby demonstrating the ability of these device arrays to illuminate specific cells,
and hence provided an initial proof-of-concept of the feasibility of using the approach for dynamic imaging from neural
circuits.
1077-260X/$20.00 © 2005 IEEE
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IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005
Fig. 1. Image of LED showing typical device geometry. Here, device diameters
are 25, 50, 100, and 200 µm.
Fig. 2. Schematic of flip-chipped LED showing basic device structure and
electrical connections, including electrical and thermal epoxies.
II. DESIGN AND LED FABRICATION
A. Choice of Wavelength
Our voltage-sensitive dye (VSD) of choice was the di-8ANEPPS (D-3167, Molecular Probes, Eugene, OR). The membrane potential-induced fluorescence change (∆F/F) of these
VSDs can be maximized by employing a combination of excitation and emission filters that do not exactly correspond with
the absorption and emission maxima of the dye. This is due
to the voltage-dependent spectral shifts that accompany the absolute fluorescence amplitude changes caused by neural cell
depolarization [8]. Although the excitation and emission peaks
of di-8-ANEPPS are at 470 and 630 nm, respectively, the best
signal-to-noise ratio with this dye could be achieved by excitation at 530 nm and fluorescence collection above 570 nm,
hence guiding the wavelength choice for our LED arrays. The
particular choice of excitation and emission filters is discussed
in Section III.
B. LED Device and Array Fabrication
The epitaxially grown p-n junction heterostructure wafers
were composed of GaN p- and n-type doped layers and an
InGaN/GaN active multiple quantum well region on sapphire
(Al2 O3 ) substrates [9]. Standard nitride device microelectronic
processing was employed to create small arrays of mesa-type
LEDs. Using a series of wet-etching and photolithographic
steps, the nitride wafer was patterned for electrical contacts
and light extraction as described next.
Following epitaxial growth, a high-temperature postgrowth
anneal was performed to activate Mg-dopants in the p-layer.
A sacrificial silicon dioxide etch mask was patterned, and a
chlorine-based reactive-ion etch tool was used to etch the nitride
to the n-type layer. A silicon dioxide passivation layer was deposited by plasma-enhanced chemical vapor deposition. Arrays
of p-electrodes, which define the optical apertures, were created by electron beam evaporation of a multilayer Ti/Al/Ti/Au
Fig. 3. Light output and current-voltage characteristics of a single 50-µm device. Output power is measured from the backside of the chip before packaging.
(100/800/100/1000 Å) metallization and lift-off technique. Several array geometries were used, with circular device apertures
ranging in diameter from 25 to 200 µm, and nearest neighbor edge-to-edge separation ranging between 25 and 150 µm
(Fig. 1). To create ohmic contacts, the material was annealed
at 600 ◦ C for 5 min in nitrogen-rich ambient. Similarly, the
n-electrode was composed of a Ni/Au (100/400 Å) metallization. Owing to the large conductivity in the n-layer, a single
n-electrode services several LED arrays in proximity, as shown
in Fig. 1. Finally, Ti/Au pads were deposited in a layout that was
convenient for electrical access and flip-chipping of the device
array onto a heat sink. Each element in the array has a unique
contact pad associated with it such that the LEDs are individually addressable and may be illuminated in any desired pattern
or time sequence (Fig. 1).
The completed arrays were flip-chipped for light extraction
through the polished sapphire backside (Fig. 2). 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
VENKATARAMANI et al.: COMPACT SEMICONDUCTOR LIGHT-EMITTING DIODES FOR DYNAMIC IMAGING OF NEURONAL CIRCUITRY
Fig. 4.
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(a) Image of packaged LED array. (b) 50-µm device ON.
(FF495-Em01) as the excitation filter to eliminate the spectral tail. The entire assembly, including the LED array, imaging optics, and excitation filter, was mounted on a micromanipulator (Sutter Instruments, Novato, CA). This arrangement enabled us to position the LED under the neural cell
chamber with practical ease. The fluorescence from the cells
was collected using a 40 × objective (Nikon E600FN microscope, Avon, MA) and, with a proper choice of dichroic
and emission filters (FF562 filter set, Semrock, Rochester,
NY), was projected onto a NeuroCCD-sm imaging camera
(RedShirt Imaging, Decatur, GA).
IV. CULTURED NEURONS
A. Cell Culture
Fig. 5.
Schematic of optical setup.
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 (Global Chip Materials,
Rancho Cordova, CA) package for easy connection to the control circuitry and mounting under the microscope.
Typical current-voltage (I–V) and optical power-current
(L–I) curves are shown in Fig. 3. The packaged LED is shown
in Fig. 4(a), and the packaged LED array with one element ON
is shown in Fig. 4(b).
III. OPTICAL DESIGN AND ENGINEERING
A schematic of the LED-based fluorescence imaging test
arrangement in shown in Fig. 5. The fluorescence collection
optics consisted of a pair of aspheric lenses (f = 5.95 mm,
F − number = 0.88). The LEDs were positioned to illuminate
the neural cells from below, and the lens pair ensured a tight focused spot at a proper distance from the target sample surface.
By carefully choosing the properties of these lenses, we were
able to achieve one-to-one focusing.
One important challenge in using LEDs as sources of
excitation is the inherent presence of the long wavelength
tail in their emission spectrum. We used a commercially
available filter set from Semrock, with a bandpass filter
The glass coverslip substrates (Carolina Biologicals, Burlington, NC) were treated in concentrated nitric acid overnight,
washed in distilled water, and then stored in 100% ethanol until further use. These coverslips were then fire polished and
placed in a 24-well plate. For regular coverslips, poly-L-lysine
(P2636, Sigma, St. Louis, MO) dissolved in borate buffer at
a concentration of 0.3 mg/mL was added and allowed to adsorb overnight. For patterning of the substrates, the coverslips
were further cleaned with acetone, methanol, and water. The
substrates were then prepared for photolithography by spinning positive photoresist (PR 1818, Shipley, Marlborough, MA)
and then exposing to ultraviolet (UV) through a chrome mask
containing the designed patterns. Upon development, patterns
of photoresist were generated on the glass substrates. poly-Llysine dissolved in borate buffer at a concentration of 1 mg/mL
was added to the coverslips and allowed to adsorb overnight.
The following day, regular coverslips were washed in distilled
water while the photoresist patterned coverslips were sonicated
in acetone to remove the unexposed photoresist, leaving predesignated patterns of poly-L-lysine on the substrate. These were
then washed with water and sterilized with 100% ethanol. Then,
DMEM (Gibco, cat# 11995065, Carlsbad, CA) 10% FBS was
added to the substrates, which were stored in the incubator (5%
CO2 , 37 ◦ C) for 2 h before plating the cells.
Cultures of rat hippocampal neurons were made as described
previously [10]. Briefly, the hippocampus was removed from
embryonic day 18 (E18) rat embryos, trypsinized (0.25%), and
dissociated by trituration. Cells were plated onto poly-l-lysinecoated glass coverslips at a density of 5000 to 10 000 cells/cm2 .
After 3 h, the DMEM/10% FBS was removed and replaced
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Fig. 6.
IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005
Top: Electrical signal from hippocampal neuron. Bottom: Optical signal using LED as excitation source.
with 37 ◦ C Neurobasal (Gibco, #21103049) media (containing Neurobasal, B27, pen-strep, and Glutamax). Cultures were
subsequently fed every four days until use.
B. Electrophysiology
Whole-cell patch-clamp recordings were performed using
an Axoclamp2B amplifier. The recording solution contained
145 mM NaCl, 3 mM KCl, 3 mM CaCl2 , 1 mM Mg Cl2 , 10 mM
Glucose, and 10 mM HEPES. The osmolality was adjusted
to 315 mmol/kg. The intracellular pipette solution contained
9 mM NaCl, 136.5 mM KGlu, 17.5 mM KCl, 0.5 mM CaCl2 , 1
mM MgCl2 , and 0.2 mM EGTA. The osmolality was adjusted
to 310 mmol/kg. pH of both the solutions was adjusted to 7.25
using KOH. All chemicals were obtained from Sigma-Aldrich
(St. Louis, MO).
C. Dye Staining
The voltage-sensitive dye that we used (di-8-ANEPPS) was
obtained from Molecular Probes. The procedure for preparing
the dye is described elsewhere [11]. Stock solutions of di-8ANEPPS were made up in DMSO at 2 mM concentration and
stored at 4 ◦ C. Cells were stained for 10 min by bath application
of 20 µm di-8-ANEPPS the bath saline. Excess dye was removed
by washing the coverslips in bath saline three times.
V. SAMPLE RESULTS
A. Procedure
As described in Section III, the LED array with its focusing
optics and the excitation filter was mounted on a micromanipulator. Similarly, the condenser of the Nikon E600FN microscope
was also mounted on a second manipulator. The cells were
stained as described previously. Using the micromanipulator,
the condenser was positioned and made to illuminate the cells
for conventional differential image contrast (DIC) viewing to
facilitate the LED alignment. After a particular cell was chosen,
an LED from the array was brought under the cell of interest, and
the coordinate position of the emitter was saved in the manipulator’s memory. Moving the LED away, the microscope condenser
was brought back in, and the cell was patched. Excitation currents were injected intracellularly, and action potentials were
generated in the target cell. Lowering the condenser, the LED
array was brought under the cell using the previously saved coordinate location. The appropriate spectral filters were brought
into position for recording the fluorescence. The fluorescence
data was acquired at 1 KHz using RedShirt CCD imaging camera. An ultralow-noise current source supplied the current to
the LED and was modulated to control the on–off timing of
the LEDs.
B. Results
A representative example of results obtained using the LEDbased excitation scheme with the optical engineering configuration described previously is shown in the raw laboratory
data of Fig. 6, focusing on the action of a single neural cell. The
top trace shows the electrical signal recorded intracellularly, displaying a single-action potential spike, whereas the bottom trace
shows the optically derived signal corresponding to modulation
in fluorescence amplitude of the voltage-sensitive dye in response to the electrical stimulus. The optical signal corresponds
to an approximately 4% change in fluorescence intensity for the
∼100 mV action potential. This data was obtained by focusing an LED to a 50-µm spot on the cell. The device was
operated at 60-mA current, illuminating the cell for a time
window of 250 ms. The LED output power level was determined to be ∼0.16 mW so the bleaching of the dye could
be minimized.
The data in Fig. 5 demonstrates the feasibility of using our
compact LED devices for voltage-sensitive dye imaging of neural cells at modest levels of excitation, when compared with the
high-power requirements in conventional optical imaging. An
LED-based illumination system thus offers a viable alternative
for fluorescent measurements of electrical activity. Considerable improvement in the signal-to-noise ratio of the modulated
VENKATARAMANI et al.: COMPACT SEMICONDUCTOR LIGHT-EMITTING DIODES FOR DYNAMIC IMAGING OF NEURONAL CIRCUITRY
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ACKNOWLEDGMENT
The authors would like to thank Dr. L. Loew (University of
Connecticut Health Center) for his guidance and useful suggestions on the VSD recordings. The authors also would like to
thank A. Taylor (Department of Neuroscience, Brown University) and Dr. J. Fallon (Professor, Department of Neuroscience,
Brown University) for their help with the neural cell culturing.
Fig. 7. (a) DIC image of hippocampal cells in culture. (b) Two of the LED
array elements under two cells. (c) Fluorescent images when both LEDs are ON
and one ON at a time.
fluorescence can be expected with further fine-tuning of the
optical and electronic subsystems. With the new capability offered by the ability of focusing individual LED illumination
onto specific cells in a patterned network, and further being able
to manipulate the pattern and time sequence of illumination,
we envision the use of these compact optical device arrays for
dynamically imaging the activity of an entire neuronal circuitry.
An initial example of this is shown in Fig. 7 for the case of for
two adjacent neurons. Fig. 7(a) shows the DIC microscope image of two such interconnected hippocampal cells. In Fig. 7(b),
two LED elements have been used to illuminate the two chosen cells. Fig. 7(c) shows series of fluorescent images of the
two cells, when both LEDs are first turned on simultaneously
and when each LED is turned on separately during a chosen
time interval.
VI. CONCLUSION
We have described and shown the applicability of using compact, planar green LEDs as effective local sources of photoexcitation for fluorescence-based imaging and applied the method
to dynamic recording of neural action in cultured hippocampal
cells. The results presented previously represent the first proofof-concept steps only, where the basic optical engineering
problems have been addressed to enable proximity illumination by individual LEDs of specific neural cells within a dense
assembly. The ability to fabricate the LEDs into planar arrays of
arbitrary spatial geometry makes the technique particularly suitable for dynamic imaging of any patterned fluorescent cellular
media. Of special interest to us are those composed of active neural cell networks. Although the choice of the LED wavelength
was here restricted to the green portion of the wavelength spectrum (dictated by the choice of a specific fluorescent dye), the
gallium-nitride-based devices can be fabricated at shorter wavelengths as well, reaching well into the sub-300-nm regime in
the UV. With the incorporation of UV LEDs, direct optical triggering of neural response becomes possible, removing the need
of the invasive intra- (or inter-) cellular electrical excitation. Finally, we note that for arrays of patterned LEDs, geometrically
matched to the multicellular landscape, the need of the (rather
expensive) imaging CCD camera is reduced. This is so because
the ability to temporally control each LED in such an array will
enable a single-element, time-gated, and synchronized photodetector to acquire full dynamic imaging information of the chosen
object scene.
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04.PDF
Sowmya Venkataramani was born in Chennai, India. She received the Sc.B.
degree in physics from Meenakshi College for Women, Chennai, in 1997, and
the Sc.M. degree in physics from Indian Institute of Technology, Madras, India,
in 1999. She received the Sc.M. degree in physics from Brown University,
Providence, RI, in 2000.
Her research interests include development of compact optical device arrays
for interactive imaging of neural circuitry.
Kristina M. Davitt was born in Canada. She received the B.Eng. degree in engineering physics from Queen’s University, Kingston, ON, Canada, in 2001 and
the Sc.M. degree in electrical engineering from Brown University, Providence,
RI, in 2004.
Her research interests include developing and characterizing UV and near
UV gallium nitride-based light emitters.
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IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 11, NO. 4, JULY/AUGUST 2005
Jiayi Zhang was born in Suzhon, China. She received the B.S. degree in physics
from Hong Kong Baptist University, Hong Kong, China, in 2003.
Her research interests include coupling of brain to micro-optoelectronic
chips.
Barry W. Connors received the Ph.D. degree in physiology and pharmacology
from Duke University, Durham, NC, in 1979.
He is currently the L. Herbert Ballou University Professor of Neuroscience
at Brown University, Providence, RI. His research interests include the cellular
and synaptic physiology of the cerebral cortex and thalamus, the properties of
electrical synapses, the behavior of small neural circuits, and the mechanisms
of epileptic seizures.
Heng Xu was born in Nanjing, China. He received the B.S. degree in physics
from Nanjing University in 2003.
His research interests include studying neural dynamics at the biophysics
interface.
Yoon-Kyu Song received the B.S. and M.S. degrees in electrical engineering
from Seoul National University, Seoul, Korea, in 1992 and 1994, respectively,
and the Ph.D. degree from Brown University, Providence, RI, in 1999.
He was a Research Scientist at Agilent Technologies (2000-03) until rejoining Brown University as an Assistant Professor (Research) in 2003. His research
interests include basic and applied semiconductor optoelectronics, such as vertical cavity lasers and nanostructured light emitters. His current research includes
work on the development of new compact UV optical sources.
Arto V. Nurmikko (M’90–SM’97–F’99) was born in Finland. He received the
B.S., M.S., and Ph.D. degrees in electrical engineering from the University of
California, Berkeley.
He is the L. Herbert Ballou University Professor of Engineering and Physics
at Brown University, Providence, RI. His current research involves basic and
device science of wide band gap semiconductors, studies of ultrafast processes
in ferromagnetically ordered systems, and high-resolution imaging of neural
circuits.
Prof. Nurmikko is a Fellow of the American Physical Society and the Optical
Society of America.