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Optically Switched Planar Microelectrode Arrays Hardware, Software

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Title: Optically Switched Planar Microelectrode Arrays Hardware, Software


1
Optically Switched Planar Microelectrode Arrays
Hardware, Software Algorithms
Tom Manuccia ProfessorDepartment of Electrical
Computer EngineeringGeorge Washington University
GWU homepage August 14, 2004
(202) 994 - 9298 manuccia_at_gwu.edu
2
Conventional (i.e., non-switched) Planar
Microelectrode Arrays
3
Chick cardiac myocytes beating on a conventional
32 electrode, non-multiplexed array in our lab
(Jan. 2002)
Because of the limited number of recording sites,
it is rare that a beating cluster of cells will
be in registry with an electrode. Three
problems limit scaling (a) The use of a single
metal layer severely constrains the routing of
traces and electrode packing density (b) One
contact pad per electrode required (c) One
pre-amp and filter per electrode required.
4
Conventional MEA (MicroElectrodeArray)
TechnologyOne Amplifier Signal Processing
Chain per Electrode
5
Multi-Layer Fabrication Solves the Routing
Other Problems
6
Multi-layer Fabrication Using BCB
  • Presently used MEA dielectrics / encapsulants
    Polyimide, Polysiloxane
  • Absorb water, swell, delaminate upon reuse
  • Poor hydrophilicity requires awkward flaming
    procedure for cell adhesion
  • Fabrication by laser ablation of individual vias
    is slow and non-reproducible
  • BCB / Cyclotene (B-staged bis(benzocyclobutene
    di-methyl siloxane) )
  • Well known planarizing, photodefinable,
    dielectric resin for semicon fabrication
  • New to MEAs, probably new to bioengineering
  • Hydrophilic but no water absorption, no
    delamination, no flaming required, non-toxic to
    neurons
  • Fabrication by conventional lithography (fast,
    high yield, etc.)
  • BCB allows multiple layer MEA designs
  • Dramatically increases total electrode count
    packing density
  • Allows for shielding layers to do stimulation and
    recording on one chip
  • Vertical stacking of traces allows optical access
    to cells from below for microscopy, absorption,
    florescence, etc.

7
Accelerated aging tests delamination, water
permeation, R, C, etc.
Major problem in field solved - ML-MEA sample
(similar construction to OSMA) after 2 weeks in
boiling saturated brine. We observed no
delamination or measurable decrease in resistance
(from many hundreds of Gigohms) between
electrodes. Other tests included adhesion (pull)
tests, strain (polarized light), pinhole
inspection, trace continuity, specific test
structures (traces crossing, interdigitated
electrodes, interlayer C, long term
biocompatability, unwanted bulk or surface
chemisorption, surface hydrophilicity /
hydrophobicity, interface Z, etc.
8
Drop-In, Pin-Compatible OEM Replacement MEA
Fabricated by Manuccias group using Multi-layer
MEA technology
9
Rat Cortical Neurons Growing on our ML-MEA
This micrograph was taken 6 days post-seeding.
This particular culture continued to thrive out
to 30 days, at which point, we recycled the plate
for durability / reuse testing. A rat
hippocampal culture continued to thrive at 52
days, with 55 out of the 64 electrodes showing
active units and S/N as large as 171. Courtesy
Ed Keefer, Neuroscience Institute, La Jolla.
10
Action Potentials Recorded with an ML - MEA
Twenty five superimposed APs from the rat
cortical neuron culture show excellent
reproducibility in amplitude, timing and shape.
Courtesy Ed Keefer, Neuroscience Institute, La
Jolla
11
Optically Controlled Electrical Switches Solves
the Connector and Parallel Electronics Problems
12
Conventional Electrical Multiplexers Cannot Be
Used
  • Conventional electrical multiplexing not adequate
    for high impedance, low voltage analog signals
    such as in this application
  • charge injection
  • voltage offsets
  • Major problem of RF interference from the 5 V
    control lines in electrical multiplexers at high
    Zs
  • With (e.g.) 10,000 on-plate, matched, low-noise,
    high gain amps, conventional analog multiplexers
    could be used, but this is cost prohibitive and
    requires opaque substrates, and/or sophisticated
    packaging.
  • Optically controlled electrical multiplexing
  • Lower charge injection artifact
  • Lower voltage offset artifact
  • Noise immunity at high switching rates
  • Optical switching completely circumvents RF EMI
  • Adaptable to extremely high packing densities

13
Optically Switched Microelectrode Array (OSMA)
Concept - I
  • One optically controlled electrical switch for
    each electrode
  • Large numbers of electrodes are connected to the
    same output bus
  • Individual electrodes are selected via low power
    laser illumination
  • For each bus, only one pad, one connector and one
    signal amplification and signal processing chain
    is needed

14
OSMA Array Concept - II
15
Early OSMA array
An early prototype OSMA 16x16 array connected to
a single output bus
16
Electrical Performance - OSMA switches
Raw data (1 mV, 1 kHz, 1 Mohm source Z) from
tests of one of our early a-Si optically
controlled switches being used to sample sine and
square waves. For many applications, slow
switches such as this would be used to select and
route only the interesting electrodes to the
output buses, and thus would stay on for the
duration of a pharmacology or other experiment.
After settling, ultimate on/off switching ratios
are in excess of 10001.
17
Noise Measurements - OSMA switches
Experimental setup and raw data Inside a
Faraday cage, an 80 microvolt simulated action
potential with 1 Megohm source impedance was
injected into one of our GaAs optically
controlled switches, and thence to a high quality
amplifier with 1 Megohm input impedance and 10 -
10,000 Hz bandwidth. The lower trace shows the
resulting signal (20 uV/div) with the laser ON.
With the laser off, the APs disappear
completely ( gt 10001), and the noise drops
slightly from 5 uV (RMS) to 4 uV (RMS). Thus,
the switches are essentially noise free when on,
and an open circuit when off. Other
measurements (not shown) included I-V curve
tracing to detect non-ohmic contacts and other
effects, photovoltaic artifacts, switching Rs
and Cs, photolinearity, time constant
measurements (carrier lifetimes/trapping),
physical adhesion, lift-off other fab flaws,
etc.
18
Complete single well 10k pixel OSMA wafer
Quadrant of an 3" wafer showing test structures
(left edge) and one complete, encapsulated OSMA
device. Contact pads are for 3 signal buses, 1
electrochemical reference bus 1 ground plane.
Structure consists of several insulating metal
layers. Because the switches are directly under
the electrodes in this design, the
electrophysiologist would use three small lasers
mounted on micropositioners as virtual
pipettes, directing them at the cells of
interest.
19
Small portion of 10k pixel OSMA
Due to lighting, the gold electrodes appear clear
in this microphotograph. 2500 of the pixels are
reference electrodes and are hard-wired to one
bus. The remaining 7500 are wired through the
switches to three signal buses.
20
Multi-well MEA Plate with OSMA Multiplexers (12
Wells, 768 electrodes)
The large wells of this plate are suitable for
slice preparations.
21
96 Well Plate Optically Switched Microelectrode
Array (6144 electrodes)
Entire electrode plate
Micrograph of one of the 96 8x8 electrode arrays
The relatively narrow wells of this plate are
suitable for cell culture work
22
Hardware Summary Multiple Generations of
Microelectrode Arrays From the P.I.s lab
Single layer, multiple layer, non-switched,
switched, amorphous-Si, GaAs, horizontal and
vertical geometry switches64 electrode OEM
drop-in array, 768 electrodes in 12 wells, 6144
electrodes in 96 wells, 10,000 electrodes in one
large (1 cm2) contiguous area, etc.
23
Environmental Chamber for 10 kPixel array
OSMA mounted in a temperature controlled
perfusion chamber on the microscope stage for
wet electrical testing. The red color is from
the pH indicator in the growth medium.
24
10 kPixel OSMA Flow / Perfusion System
25
Data Analysis and Simulation
26
Data Analysis Software - Ensembles
  • Designed to analyze data from large electrode
    count MEAs in real or near real time.
  • Detects and quantifies small changes in 1st and
    2nd order statistics of the firing patterns.
  • Detects and enumerates membership in
    correlationally defined neural ensembles.
  • Detects subtle phase changes in firing patterns
    without any change in rates, ISIs, etc..
  • Left - Color coded cross correlogram in a
    simulated 100 neuron network exhibiting chaotic
    firing. The weights of all connections crossing
    a cut plane have been reduced.
  • Right - Correlogram of the same data after
    re-indexing of the neuron IDs by Ensembles
  • The presence of two distinct ensembles is obvious
    (i.e., two diagonal blocks).

27
Example of Another Use of Ensembles Detecting
the Change Between 1 and 2 Ensemble Behavior In a
Network With a Soft Cut-Plane
  • A quasi-chaotically firing, randomly connected
    network was modified by multiplying the synaptic
    weights of all processes crossing a cut-plane.
    The resulting spike time data was fed to
    Ensembles for analysis. With weak coupling, the
    two sub-nets fire independently. As the coupling
    increases, activity in the sub-nets becomes more
    correlated.
  • The above graph shows the variation of the firing
    similarity metric for numerous pairs of neurons
    as a function of coupling strength. With weak
    coupling, the similarities clearly cluster into
    two groups, same-side and opposite-side, but
    start to merge at coupling strengths as low as
    10-4. For stronger coupling, the networks
    effectively act as one.

28
Data Simulation Software - NeuroSpike
Can simulate 100,000 biologically realistic
neurons on a desktop PC. Includes neuron level
rate adaptation, spike timing dependent synaptic
plasticity, statistical distribution of neuron
location, orientation, and many other parameters.
Used to simulate streaming data from high
electrode count MEAs for input to Ensembles.
May also have AI / ANN interest. Uses an
optimistic discrete event simulation kernel.
29
Data Simulation Software - NeuroSpike
Typical data generated by NeuroSpike. This shows
the activity of the inhibitory neurons in a
simulation of 1000 randomly connected, randomly
located simple integrate and fire neurons (900
excitatory, 100 inhibitory). Different
parameters can generate a rich variety of
behaviors including epileptic-like
spatio-temporal waves of activity, clusters of
cells (ie, ensembles) acting in concert, but only
weakly connected to other clusters, network wide
bursting behavior, etc.
30
Major Achievements
  • First microelectrode arrays with multiple layer
    construction
  • Overcomes trace routing problems. Allows for 10X
    - 100X increase in electrode count
  • Introduction of a new, biocompatible insulating
    material with vastly improved physical and
    chemical properties.
  • Overcomes previous durability and cell adhesion
    problems.
  • First microelectrode array incorporating an
    optically controlled electrical multiplexer
  • Overcomes previous scaling limitations. Allows
    reasonable number of connections and amplifier
    chains. Can select only those electrodes of
    interest. Glass substrate.
  • First microelectrode array with more than 1 or 2
    wells
  • MEAs no longer just for basic research.
    Sequential experiments washout no longer
    required.
  • First software simulator of biologically
    realistic neuronal networks that can handle
    networks of 105 neurons on a desktop machine at
    reasonable speed
  • Important as gold-standard data source for data
    analysis programs designed to handle the data
    from large numbers of neurons simultaneously (ie,
    advanced MEAs)
  • First data analysis software to detect changes in
    ensemble firing behavior in data streamed from
    large electrode count MEAs.
  • Critical to high throughput, high sensitivity,
    high selectivity assays

31
Selected formal and collegial relationships -
Academia Neuroscience Institute (La Jolla) Dr.
Ed Keefer performing tests on our arrays will
use them in his ongoing research efforts,
probable joint grant applications, joint
papers. Univ. North Texas Prof.. Guenter Gross
performing tests on our arrays, supplied field
potential data from his cell culture work for our
data analysis efforts. Case Western Reserve
Univ. Medical School Prof. Bryan Roth head of
NIMH psychoactive drug screening program
committed to use of our system, possible joint
grant applications. Pittsburgh Supercomputing
Center Discussions with facility director in
regards to parallelizing Ensembles for very large
on-line data analysis tasks (i.e., High
Throughput Screening). Suggested that obtaining
grant support would be easy. Penn State Dr.
Jeffrey Catchmark Associate director, co-author
of one patent application. The NSF
Nanofabrication Facility at Penn State was used
for most fabrication tasks. Howard Univ. Dr.
Gary Harris The NSF Nanofabrication Facility at
Howard was used for occasional fabrication tasks.
32
Selected formal and collegial relationships -
Government NIMH Drs. Mike Huerta, Dennis
Glanzman Program managers for our efforts for
many years NIMH Dr. Linda Brady Head NIMH
psychoactive drug screening program - Strongly
supports the potential HTS application of our
technologies. NINDS Dr. Bill Heetderks (now
NIBIB) Program manager for our multi-layer
fabrication advance. Naval Research Laboratory
Dr. Joe Pancrazio Performed initial testing on
our arrays, probable collaborator in future joint
efforts, papers. Wants to use our arrays. DARPA
Dr. Alan Rudolph DoDs Tissue Based
Biosensors Program interested in HTS
applications of this technology
33
Selected formal and collegial relationships -
Commercial Merck Drs. Jeff Conn head of
Neuroscience Setting requirements for HTS
system, possible RD support, collaborative
efforts. Lilly - Dr. Gary Tollifson - head of
Neuroscience products - Interested in our system
for slice preparations. Tranzyme, Inc. Dr.
Ramabhadran Collaboration / system purchase for
studies of transfected neurons Applied Neuronal
Network Dynamics, Inc. Mr. Daron Evans
Customer for drop-in replacement arrays Research
International Dr. Elric Saaski Teaming
partner to engineer the environmental and thermal
control subsystems. Tensor Biosciences Dr.
Miro Pastrnak Possible teaming partner slice
preps Loftstrand, Inc. Dr. Pat Manos
Performed tests with cardiac myocytes in our
lab. SAIC Dr. Paul Schaudies Possible
chem/bio warfare applications. NeuraLynx, Inc.
C. Stengel Teaming partner to supply data
acquisition hardware, possible distributor of our
software for basic neuroscience users,
independent of HTS - drug discovery
neurotoxicology applications.
34
Related Grant Support (taken from NIH CRISP)
Grant Number PI Name Project Title 1r43dc004480-
01 Manuccia, Thomas NeuroSpike Software For The
Simulation Of Neuronal Networks 1r43mh052010-01 Ma
nuccia, Thomas Initiated-Event Model Of
Statistical Point Processes 1r43mh052977-01 Manucc
ia, Thomas Processing And Display Of Correlations
In Multineuron Events 1r43mh054410-01a1 Manuccia,
Thomas Optically Switched Microelectrode
Array 1r43mh059442-01 Manuccia, Thomas Software
For The Detection Of Neural Ensembles 1r43ns04392
9-01 Manuccia, Thomas Technology for Ultra-dense
Microelectrode Arrays 2r44mh052010-02a1 Manuccia,
Thomas Initiated Event Models Of Stochastic
Point Processes 5r44mh052010-03 Manuccia,
Thomas Initiated Event Models Of Stochastic Point
Processes 2r44mh054410-02 Manuccia,
Thomas Optically Switched Microelectrode
Array 5r44mh054410-03 Manuccia, Thomas Optically
Switched Microelectrode Array 5r44mh054410-04 Man
uccia, Thomas Optically Switched Microelectrode
Array 3r44mh054410-04s1 Manuccia,
Thomas Optically Switched Microelectrode
Array 2r44mh059442-02a2 Manuccia, Thomas System
For The Detection Of Neural Ensembles 5r44mh05944
2-03 Manuccia, Thomas System For The Detection Of
Neural Ensembles
35
Sampling of Other Biomedical Engineering and
Sensing Projects From the Lab of the P.I.
1. Coherent Anti-Stokes Raman Microscopy - Allows
chemical species selective spatial visualization
- e.g., the distribution of lipids, deuterated
lipids, etc. within cells. Essentially a mid-IR
microscope with the spatial resolution of a
visible light microscope. 2. Laser Electron
Microscope - Similar to 1. Essentially a mid-IR
microscope with the spatial resolution of a
scanning electron microscope. 3. Mass transport
to/from coated droplets acoustically levitated in
a free-jet wind tunnel. Possible chem/bio
warfare utility.4. Ultrasensitive and selective
(ie, sub-ppt) detection of NO by Zeman modulated
acousto-optic detection. Application to
explosives detection.5. Production and imaging
of ultrasound by pulsed RF in tissue - Contrast
production by spatially varying dielectric
material properties not acoustic properties.
Application to the detection of breast
malignancies.
36
Staff of the Schafer BioEngineering Group - Dec.
20, 2002 (Missing Pat Manos, Ingrid Mahogony
--- Includes the P.I.s wife and daughter)
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