U.S. patent application number 15/041589 was filed with the patent office on 2016-06-09 for multiwell microelectrode array with optical stimulation.
The applicant listed for this patent is Axion BioSystems, Inc.. Invention is credited to Isaac Perry Clements, Daniel Christopher Millard, Amanda Jervis Preyer, Swaminathan Rajaraman, James David Ross.
Application Number | 20160161465 15/041589 |
Document ID | / |
Family ID | 54834460 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160161465 |
Kind Code |
A1 |
Clements; Isaac Perry ; et
al. |
June 9, 2016 |
MULTIWELL MICROELECTRODE ARRAY WITH OPTICAL STIMULATION
Abstract
An electro-optical stimulation and recording system is
disclosed, including a substrate and a plurality of wells coupled
to the substrate. The system also includes at least one electrode
set disposed proximate a respective one of the plurality of wells,
wherein the electrode set comprises at least one electrode
configured to collect an electric signal associated with at least a
portion of the respective well. The system also includes a
light-emitting element set corresponding to a respective one of the
wells and configured to deliver optical stimulation to at least a
portion of the respective well.
Inventors: |
Clements; Isaac Perry;
(Marietta, GA) ; Preyer; Amanda Jervis; (Atlanta,
GA) ; Rajaraman; Swaminathan; (Decatur, GA) ;
Millard; Daniel Christopher; (Atlanta, GA) ; Ross;
James David; (Decatur, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Axion BioSystems, Inc. |
Atlanta |
GA |
US |
|
|
Family ID: |
54834460 |
Appl. No.: |
15/041589 |
Filed: |
February 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14738618 |
Jun 12, 2015 |
9279797 |
|
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15041589 |
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62011291 |
Jun 12, 2014 |
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Current U.S.
Class: |
506/39 |
Current CPC
Class: |
B01L 2300/0829 20130101;
G01N 21/75 20130101; G01N 2201/062 20130101; G01N 21/01 20130101;
B01L 2300/0645 20130101; B01L 2300/0654 20130101; G01N 2201/0446
20130101; G01N 2021/0346 20130101; G01N 21/64 20130101; B01L
3/50853 20130101; G01N 2021/015 20130101; B01L 2300/046 20130101;
G01N 33/4836 20130101 |
International
Class: |
G01N 33/483 20060101
G01N033/483; G01N 21/01 20060101 G01N021/01 |
Claims
1-28. (canceled)
29. An optical stimulation system, comprising: a microplate
comprising a plurality of wells; a lid configured to couple to the
microplate, wherein at least a portion of the lid contacts the
microplate; and at least one light-emitting element set
corresponding to at least one of the plurality of wells and
configured to deliver optical stimulation to the at least one well
of the plurality of wells, wherein the at least one light-emitting
element set is configured to be removably coupled to the lid, and
wherein the lid is configured to modify an optical property of
delivered light.
30. The system of claim 29, wherein the microplate is configured to
enhance light delivery within each of the plurality of wells or to
reduce light bleed-through between two or more of the plurality of
wells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/738,618, filed on Jun. 12, 2015, entitled
"MULTIWELL MICROELECTRODE ARRAY WITH OPTICAL STIMULATION," which
claims the benefit of U.S. Provisional Patent Application No.
62/011,291, filed on Jun. 12, 2014, entitled "MULTIWELL
MICROELECTRODE ARRAY WITH OPTICAL STIMULATION," the disclosures of
which are expressly incorporated herein by reference in their
entireties.
TECHNICAL FIELD
[0002] The present disclosure relates generally to tools for
facilitating optogenetic stimulation of cells and tissue and, more
particularly, to multiwell microelectrode arrays with optical
stimulation capabilities and associated methods for using the
same.
BACKGROUND
[0003] Microelectrode arrays (MEAs) are an invaluable tool for
scientific discovery and medical research. Because they can
actively monitor and manipulate cellular activity (at both the
single-cell and tissue levels) using electrical
stimulation/recording, MEAs provide extraordinary insight into cell
network interactions. Many conventional MEAs are of the single-well
variety, meaning that only a single cell or tissue culture may be
tested/analyzed at a time. Consequently, testing of multiple cell
or tissue samples using conventional single-well microplates
typically requires a significant monetary investment in multiple
single-well measurement test beds, a significant allocation of time
to sequentially test each cell or tissue sample, or some
combination of the two.
[0004] To provide a more cost- and time-efficient platform for
simultaneously testing multiple cell or tissue cultures, multiwell
MEAs were developed. Unlike their single-well counterparts,
multiwell MEAs provide an array of culture wells, each of which has
a corresponding array of electrodes for recording electrical
activity from (and/or delivering electrical stimulation to) the
contents of the well. Current multiwell MEAs come in a variety of
sizes, including, 4-, 12-, 24-, 48-, 72-, 96-, and 384-well
configurations, providing a significant number of options for
scaling in vitro testing to meet the needs of most any experimental
setting. Although multiwell MEAs have certainly alleviated the
scalability problems associated with single-well MEAs, they are
generally limited in their ability to deliver different modes of
stimulation (e.g., electrical, optical, thermal, etc.)
[0005] More specifically, despite the relative success of multiwell
MEA systems, the technology's impact may be limited by the inherent
limitations of electrical stimulation. Electrical stimulation
pulses from MEA microelectrodes are limited to the locations of the
electrodes and excite all nearby electroactive cells, regardless of
cell sub-type. Electrically mediated inhibition of cell activity
requires complex stimulation paradigms that are impractical and
unreliable. Additionally, the amount of charge injection required
for extracellular stimulation can saturate sensitive electronics
and leave residual charge on the electrodes. In turn, this charge
creates blind spots in electrical recordings that obscure critical
activity around the time of stimulation. Therefore, there is a need
for new stimulation solutions that can more selectively control
cell networks without creating distortions or artifacts in the
electrophysiological recordings.
[0006] Optogenetic stimulation techniques provide a more selective
mechanism for manipulating cell cultures. In optogenetics
methodologies, selected cells are genetically manipulated to
express light sensitive membrane proteins called opsins. Specific
cell types within heterogeneous cultures can then be genetically
targeted for activation or inhibition with light of specific
wavelengths. This light can be precisely pulsed and more evenly
delivered across cultures, stimulating (or inhibiting) only the
targeted cell types, while creating minimal stimulation artifact.
Using different methodologies, optogenetic stimulation can
alternatively provide the capability to influence intracellular
signaling.
[0007] In order to provide a multiwell MEA solution with enhanced
capability for selectively targeting different types of cells
within a culture or tissue sample, a multiwell MEA system with
integrated, independently controllable optical stimulation
capabilities would be advantageous. The presently disclosed
multiwell microelectrode arrays with integrated optical stimulation
capabilities and associated methods for using the same are directed
to overcoming one or more of the problems set forth above and/or
other problems in the art.
SUMMARY
[0008] According to one aspect, the present disclosure is directed
to an electro-optical stimulation and recording system, comprising
a substrate and a plurality of wells coupled to the substrate. The
multiwell plate may also include one or more electrode sets, each
electrode set disposed proximate a respective one of the plurality
of wells. Each electrode set comprises at least one electrode
configured to collect an electric signal associated with at least a
portion of the respective well. The multiwell plate may also
include at least one light-emitting element set corresponding to a
respective one of the wells and configured to deliver optical
stimulation to at least a portion of the respective well.
[0009] In accordance with another aspect, the present disclosure is
directed to a method for large-scale in vitro testing or
manipulation of cell cultures. The method may comprise providing a
control signal for causing a light-emitting element to emit light
in an illumination pattern, the light-emitting element disposed
proximate a well of a multiwell plate. The method may also comprise
detecting, via an electrode disposed proximate the well, a signal
associated with at least a portion of the well. The method may also
comprise analyzing the detected signal and outputting information
indicative of the analysis. The method may also comprise comparing
the detected signal with benchmark data. The method may also
comprise determining, based on the comparison, that adjustment of
the illumination pattern is required. The method may also comprise
modifying one the illumination pattern based on the determination.
The detected signals may be analyzed, and information indicative of
the analysis may be output, via a user interface element on a
display. Optical stimulation parameters may be automatically
adjusted based on detected signals, on a per well basis. Such
automatic, algorithmic adjustment may be useful, for example, to
optimally adjust light patterns on a per well basis. Software tools
may allow light patterns to be generated and directed to selected
wells and provide visualization of delivered light alongside
visualizations of detected electrical activity.
[0010] In accordance with another aspect, the present disclosure is
directed to a an electro-optical stimulation and recording system,
comprising an electrode set disposed proximate a first well and
comprising a first electrode configured to collect an electric
signal associated with at least a portion of the first well. The
system may also comprise a first light-emitting element set
configured to deliver optical stimulation to at least a portion of
the first well; and a processor. The processor may be configured to
provide a first control signal for causing the first light-emitting
element to emit light at a first illumination pattern; and detect,
via the first electrode, a first signal associated with at least a
portion of the respective well.
[0011] In another example embodiment, the present disclosure is
directed to an optical stimulation system comprising a microplate
having a plurality of well. The system may also comprise at least
one light-emitting element set corresponding to at least one of the
plurality of wells and configured to deliver optical stimulation to
at the at least one well. The system may also comprise a lid
configured to couple to the microplate, wherein the lid enhances
delivery of light through the lid via at least one of recesses,
lenses, and reflective surfaces.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates an example system having a multiwell
microelectrode array with optical stimulation delivery capability
for controllable stimulation of culture wells, in accordance with
certain disclosed embodiments.
[0013] FIG. 2 provides a detail view of an optical stimulation
control and delivery portion of the system illustrated in FIG. 1,
consistent with certain disclosed embodiments.
[0014] FIG. 3A provides a diagrammatic perspective view of an
example 48-well microelectrode array (MEA), consistent with certain
disclosed embodiments.
[0015] FIG. 3B provides a zoomed diagrammatic perspective view of a
selection of wells of a 48-well microelectrode array (MEA), in
accordance with certain disclosed embodiments.
[0016] FIG. 4 provides a diagrammatic perspective view of a
multiwell microelectrode array (MEA) with a detachable optical
stimulation module coupled to the MEA, consistent with certain
disclosed embodiments.
[0017] FIG. 5 provides a schematic block diagram of a cross-section
of a multiwell microelectrode array (MEA) with a detachable optical
stimulation module coupled to the MEA, in accordance with certain
disclosed embodiments.
[0018] FIG. 6 provides a schematic block diagram of a cross-section
of another embodiment of multiwell microelectrode array (MEA) with
a detachable optical stimulation module coupled to the MEA,
consistent with certain disclosed embodiments.
[0019] FIG. 7A provides an overhead (top) view of an exemplary
single well (i.e., corresponding to a single culture well of a
multiwell MEA) 8.times.8 grid of electrodes used in the presently
disclosed multiwell microelectrode array (MEA) with optical
stimulation capabilities, in accordance with certain disclosed
embodiments.
[0020] FIGS. 7B and 7C provide detail overhead (top) zoom views of
an exemplary single (i.e., corresponding to a single culture well
of the multiwell MEA) 8-microelectrode array used in the presently
disclosed multiwell microelectrode array (MEA) with optical
stimulation capabilities, consistent with certain disclosed
embodiments.
[0021] FIG. 8 provides a block diagram of a processor-based
computing system having an integrated data analysis and control
suite that is used to control and conduct various experiments using
the presently disclosed multiwell microelectrode array (MEA) with
optical stimulation capabilities, in accordance with certain
disclosed embodiments.
[0022] FIGS. 9A, 9B, 9C, 9D, and 9E provide screen shots associated
with an example user interface associated with the integrated data
analysis and control suite that is used to conduct and visualize
various experiments that may be performed using the presently
disclosed multiwell microelectrode array (MEA) with optical
stimulation capabilities, consistent with certain disclosed
embodiments.
[0023] FIG. 10 illustrates an exemplary multiwell microelectrode
array system containing a 48-well multiwell microelectrode array
plate, and a detachable optical stimulation device for optically
stimulating individual culture wells, in accordance with certain
disclosed embodiments.
[0024] FIG. 11 provides a schematic block diagram of certain
components associated with the exemplary interface system
illustrated in FIG. 10 and its corresponding interactions between
the multiwell MEA plate 300 and control and monitoring system
800.
[0025] FIG. 12 provides a functional block diagram associated with
an exemplary process for use and control of the presently disclosed
multiwell microelectrode array (MEA) with optical stimulation
capabilities, consistent with certain disclosed embodiments.
[0026] FIG. 13 provides a diagrammatic perspective view of an
individual culture well within an exemplary multiwell
microelectrode array (MEA) plate, along with an overlying
detachable optical stimulation module, consistent with certain
disclosed embodiments.
[0027] FIGS. 14A, 14B, and 14C provide diagrammatic cross-sections
of individual culture wells in different example multiwell
microelectrode array (MEA) plates, along with overlying detachable
optical stimulation modules, shown coupled to the MEA plates. The
multiwell MEA plate lids and LED reflectors in FIGS. 14A, 14B, and
14C are specialized to enhance optical delivery and/or prevent the
passage of light between adjacent wells, consistent with certain
disclosed embodiments.
DETAILED DESCRIPTION
[0028] FIG. 1 illustrates an exemplary electro-optical stimulation
delivery system 100 having a multiwell microelectrode array and
optical stimulation delivery capability in accordance with certain
disclosed embodiments. The electro-optical stimulation delivery
system 100 illustrated in FIG. 1 may provide a platform for
controlling delivery of optical (and, in some cases, electrical)
stimulation to a subset of culture wells and actively monitoring
the electrical activity associated with the cells contained in the
well. The electro-optical stimulation delivery system 100 may
include, among other things, an optical stimulation module 210, a
multiwell microelectrode array 300, and an MEA plate interface
1000.
[0029] Optical stimulation module 210 is also referred to herein as
"optical stimulation device." In some embodiments, an optical
stimulation module may be integrated with the MEA plate. In other
example embodiments, an optical stimulation module may be a
standalone device that detachably couples to an MEA plate. For
example, the optical stimulation module may couple to the top of
the MEA plate or may be coupled to the bottom of the MEA plate.
Other configurations are possible as well and the language herein
is not intended to limit the possible implementations of the
optical stimulation module.
[0030] Those skilled in the art will recognize that the components
described above with respect to electro-optical stimulation
delivery system 100 are exemplary only and not intended to be
limiting. Consequently, electro-optical stimulation delivery system
100 may include additional, and/or different components than those
shown in FIG. 1 without departing from the scope of the present
disclosure. Each of the individual components of electro-optical
stimulation delivery system 100 shown in the embodiment illustrated
in FIG. 1 will be described in greater detail below.
[0031] Although FIG. 1 illustrates optical stimulation module 210
and multiwell MEA plate 300 as separate elements, it is
contemplated that one or more of the features of optical
stimulation module 210 may be included as part of multiwell MEA
plate 300 (or vice versa). Indeed, certain embodiments contemplate
a multiwell microelectrode array with integrated optical
stimulation capabilities, regardless of whether the optical
stimulation capabilities are provided by a separate, standalone
system (as shown in FIG. 1), or by an integrated solution as part
of a multiwell MEA plate 300.
[0032] FIG. 2 illustrates a schematic block diagram of an optical
stimulation delivery system 200. Optical stimulation delivery
system 200 includes a plurality of components that control the
delivery of optical stimulation to MEA plate 300. As illustrated in
FIG. 2, and in accordance with one embodiment, optical stimulation
delivery system 200 may include an optical stimulation module 210
and a control and monitoring system 800 that is programmed to
control optical stimulation module 210.
[0033] Optical stimulation module 210 may include one or more
light-emitting element sets 230 that may be optimized/customized to
meet most any power and thermal criteria that may be required.
Optical stimulation module 210 may also include control circuitry
220 for independently controlling the light-emitting elements with
light patterns of high temporal resolution, as well as finely
graded control over output intensity. The system may also include
interactive software that will enable generation of customizable
stimulation waveforms, and visualization of these delivered
stimulation waveforms. As such, the presently disclosed system may
be configured to manage cell-targeted activation or inhibition for
multiple cultures in a multiwell MEA. The term "light-emitting
element set" is not intended to limit the number of light-emitting
elements within a given "set." For example, a light-emitting
element set may comprise one, or multiple, light-emitting
elements.
[0034] As shown in FIG. 2, each of light-emitting element sets 230
may include or embody any suitable component for delivering optical
energy to a respective culture well with which the light-emitting
element is associated. According to one embodiment, (and as
illustrated in FIG. 4), light emitting diodes (LEDs) may be
provided in an overlying or lid-based array (at least one LED per
well). This configuration is particularly advantageous in
situations where a compact, top-side form factor may be required.
Importantly, however, although certain embodiments call for a
single LED per well, it is certainly contemplated that
light-emitting element sets may include multiple light-emitting
elements per well (as illustrated, for example, in FIG. 6). It is
contemplated that, although LEDs are described in certain exemplary
embodiments as the light-emitting elements, any of a number of
light-emitting sources may be adapted for implementation. For
example, light-emitting elements may include or embody any (or a
combination) of lasers, organic light-emitting diodes (OLEDs), 2D
pixel displays, digital light projection (DLP) technology, a
waveguide element, a light-emitting carbon nanotube, or any other
element suitable for providing a controllable light source for
delivering optical energy to the culture well.
[0035] As illustrated in FIG. 2, each light-emitting element 230
may be coupled to control and monitoring system 800 via control
circuitry 220. Light-emitting elements 230 may be controlled by any
of a number of commercially available control modules. In
embodiments that implement LEDs as the light-emitting elements,
current-control modules (e.g., RCD24-1.0, RECOM) may be used. Such
circuits may be used to control high intensity LEDs (e.g., Oslon
SSL, Osram Opto Semiconductors) of one or more wavelengths.
Light-emitting elements 230 may be configured to deliver one or
more of infrared light energy, ultraviolet light energy, or visible
light energy, depending upon the type of experiment being performed
(and/or the type of light-reactive opsin being targeted, in the
case of optogenetics applications).
[0036] Control circuitry 220 for the optical stimulation device may
be configured to independently control individual light-emitting
elements or may control sets of multiple light-emitting elements as
a group. System control may be handled by a 32-bit microcontroller
(e.g., MCF 5213; Freescale). Each LED in the array might be driven
by an RCD24-1.0 LED Driver. This driver may control LED intensity
with an analog input signal, which may be set by the
microcontroller through a high channel count digital to analog
converter (AD5391, Analog Devices). The driver may also take a
digital on/off signal for pulsed or triggered waveforms. The
microcontroller may be programmed via a high speed USB-to-UART/FIFO
(FT2232H, FTDI) chip.
[0037] According to an exemplary embodiment, light-emitting
elements 230 may be positioned above individual wells on an n-well
MEA plate and driven continuously or modulated at a fixed or
variable current during electrical recordings. The presently
disclosed multiwell microelectrode array with coupled optical
stimulation module may be configured to reduce both steady-state
and transient noise caused by the light-emitting elements.
Steady-state electrical noise may be eliminated with proper
grounding of the LED driver circuitry. Electrically-induced
transient noise may be reduced/eliminated by placing a transparent,
electrically conductive, grounded layer (e.g., indium tin oxide
(ITO)) between the LED and MEA well. When present, any
photoelectric artifact may be reduced with, for example, typical
bandpass filtering used for recordings of electroactive cells (e.g.
200-3000 Hz bandpass filter for spike signals (e.g., neural or
cardiac)).
[0038] As explained, electro-optical stimulation delivery system
100 comprises one or more components that cooperate to deliver
optical (and, in some embodiments, electrical) stimulation to one
or more of a plurality of culture wells in a multiwell
microelectrode array device 300 and simultaneously detect
electrical signals from the culture wells, including those signals
that are indicative of the cellular response to the stimulation
FIGS. 3A and 3B each provide a diagrammatic perspective view of the
structural components of a multiwell culture array 300. As
illustrated in FIGS. 3A and 3B, a multiwell culture device
(regardless of whether it is configured as a multiwell MEA or
multiwell MEA with optical stimulation capabilities) typically
includes a plurality of culture wells 310 disposed upon substrate
312. FIG. 3A illustrates multiwell array 300 as a 48-well device.
It is contemplated, however, that any number of wells may be
provided without departing from the scope of the present
disclosure. Furthermore, the dimensions shown in FIG. 3A are
exemplary only, and not intended to be limiting. According to an
exemplary embodiment, the presently disclosed multiwell arrays may
be sized and configured in an ANSI-SLAS compliant format,
compatible with traditional plate readers and automated
instrumentation.
[0039] Multiwell MEA 300 includes a matching lid designed for
maintaining sterility and reducing media evaporation. Each culture
well comprises an internal volume for receiving cell culture
material at an opening in the top of the well. In one embodiment,
cells can be added within a small drop to the center of the well
(where the MEA electrodes reside). After the cells have an
opportunity to attach to the substrate, then the well can be
flooded with more cell culture medium. Electrical signals resulting
from cellular reactions can be monitored by electrodes positioned
in the bottom of culture well. Electrical connections for
connecting the electrodes to MEA plate interface 1000 are located
on the bottom-side of the device. It is contemplated that the
multiwell MEA might be used along with other biological,
environmental, or chemical samples aside from cell cultures.
[0040] As shown in FIG. 3B, each culture well 310 may embody a
receptacle for receiving cell culture and/or tissue material
through an opening at the top of the well. According to one
embodiment, the walls 311 and/or substrate and/or lid of culture
well 310 may be comprised of or coated with reflective material to
maximize the light delivery to the base of the well and to prevent
light absorption or loss through the wall of the well. According to
one embodiment, reflective white-walled multiwell plates may be
used to increase light delivery by reflecting and concentrating
light within the wells, while blocking passage of light to adjacent
wells. Additionally, light delivery may be increased through the
use of commercially available clear media, as culture media dyed
with Phenol Red was found to significantly absorb blue and green
light. Electrodes (not shown) positioned at the base 312 of the
culture well 310 can be configured to provide electrical
stimulation and monitor any electrical cellular, biological, or
chemical activity or cellular, biological, or chemical sample
properties within a given well.
[0041] FIGS. 4, 5, and 6 illustrate an exemplary configuration of
the multiwell microelectrode array 300 with coupled optical
stimulation module 210 consistent with the disclosed embodiments.
Typical mid- to high-power LEDs can draw high current loads and
dissipate significant heat. For example, a high power 465 nm blue
LED might consume 1 W power and dissipate 0.5 W of heat when run at
a moderate supply current of 350 mA. These types of power and
thermal costs scale with LED count, required light intensity, and
stimulation duty cycle. Consequently, the presently disclosed
system includes robust power supply circuitry 410 with dedicated
control of the current delivered to each LED (230A, 230B, 230N). As
shown in FIGS. 4 and 5, multiwell MEA 300 may include one or more
components for managing heat dissipation to ensure efficient LED
operation, prevent LED device damage, and reduce the amount of heat
delivered to cell cultures. The system may utilize metal core
printed circuit boards and, in some embodiments, a custom aluminum
or copper heat sink 420 with an optional active cooling system.
Although FIGS. 4 and 5 illustrate the optical stimulation module
210 having a single LED per set, it is contemplated that each set
may include a plurality of independently controllable light
emitting elements 650 configured to deliver multiple wavelengths of
light to each well.
[0042] FIG. 6 illustrates an exemplary multiwell microelectrode
array with a coupled optical stimulation module, where each
light-emitting element set includes a plurality of light-emitting
components 650. Each light-emitting component may be configured to
deliver a different wavelength and/or intensity of light to provide
greater flexibility in the types of experiments that can be
performed. In addition to multiple light-emitting elements, the
multiwell MEA 300 may also include one or more devices for
enhancing the delivery of optical energy to the culture well. For
example, as illustrated in FIG. 6, multiwell MEA 300 may include
one or more light-collimating reflectors 640 for directing the
optical energy to the center of the culture well. Alternatively or
additionally, multiwell MEA 300 may also include other components,
such as lenses, filters, gratings, and other components for
modifying the optical properties depending upon the specific needs
of the experiment. These features might be incorporated into the
lid 610 of the multiwell plate. For example, lenses and recesses
can be inexpensively molded into a low-cost disposable multiwell
plate lid.
[0043] As also illustrated in FIG. 6, the optical stimulation
module 210 may be configured to overlay a transparent lid 610
associated with the multiwell MEA 300. This configuration may allow
the optical stimulation module 210 to be coupled and de-coupled
from multiwell MEA 300, without potentially contaminating the
cultures during testing. In this configuration, optical stimulation
module 210 may be designed to removably couple to/from lid 610. As
an alternative to the optical stimulation module 210 being
removably coupled to a separate lid, it is contemplated that
optical stimulation module 210 may be integrated with lid 610, or
constructed to be the lid for the multiwell MEA 300.
[0044] Although FIG. 6 illustrates light-emitting elements that are
configured for positioning at the opening of (i.e., above)
multiwell MEA 300, it is contemplated that the light-emitting
elements may be embedded in a circuit board beneath a transparent
floor of the respective wells. According to one exemplary
embodiment, light emitting elements may be provided beneath or
within substrate 620 upon which the grid of electrodes is disposed.
In this embodiment, the electrodes may be printed on a transparent
substrate (e.g., Printed Circuit Board (PCB), flex circuit, or
transparent biosensor array) through which light can pass.
According to other embodiments, light emitting elements may be
included as part of the same transparent substrates upon which
electrodes are disposed. Organic Light Emitting Diode (OLED)
technology can be coupled with micro-biosensor fabrication
technologies to achieve such an integrated transparent OLED-MEA
substrate.
[0045] According to additional and/or different embodiments, the
multiwell MEA 300 may also include enhancements to facilitate
efficient conservation/delivery of light emitted by optical
stimulation module 210. For example, multiwell MEA 300 may include
strategically shaped or curved walls 660 to ensure more
concentrated light delivery toward the central portion of the
bottom of the well. Alternatively or additionally, multiwell MEA
may include a reflective material 630, disposed beneath each well
and configured to reflect light that is transmitted through the
bottom of the well back into the well (in the case of a transparent
substrate 620). The MEA lid 610 may also incorporate modifications
to increase, enhance, shape, or otherwise influence light delivery
to the MEA culture. For example, lenses might be molded into the
lid to collimate or refract light to the center of the well. A
Fresnel lens design might accomplish this while extending minimally
into the well. The light might have recesses to allow light sources
such as LEDs or optical fibers to extend some depth into the well,
increasing light delivery, or directing light to one or more
particular regions of the culture. To minimize condensation that
might collect on the lid in the course of typical use, possibly
interfering with light delivery, a system of heating the lid might
be employed. In one example, a transparent ITO layer, might be used
to heat the lid and minimize condensation. In the case of
bottom-side light delivery, the lid might be reflective, to
re-direct and concentrate light back into the culture well.
[0046] As explained, multiwell MEA 300 includes one or more
electrodes, each of which is configured to measure electrical
activity in the surrounding area. Multiwell MEA 300 includes one or
more electrode sets, each electrode set comprising one or more
(e.g., 8) electrodes, with each set disposed between the substrate
312 and the base of a respective culture well that it is configured
to monitor. The term "electrode set" is not meant to limit the
number of electrodes in the "set." For example, an electrode set
may comprise a single electrode or multiple electrodes.
[0047] FIG. 7A illustrates a schematic of an exemplary electrode
array. As shown in FIG. 7A, electrode arrays are made up of a grid
of tightly spaced electrodes, and each electrode is capable of
simultaneously monitoring the activity of individual cells. As
illustrated in FIG. 7A, the arrangement of multiple electrodes in a
grid extends the recording range across a relatively large area,
providing concurrent access to both single cell and tissue- or
network-level activity. The control and monitoring of this cellular
activity is made possible by the electronics, which impart multiple
functions to each electrode.
[0048] Each electrode of the array facilitates monitoring of
single-cell and network-level activity for extended periods of
time, with virtually no destructive interference to the tissue
being investigated. In fact, the broad access to network
information, along with the minimally invasive nature of the
device, is precisely what makes the MEA an exceptional single-cell
and network-level research tool. Each electrode in the high
throughput MEA is ideally suited for investigation of electroactive
cells and tissue (e.g., neural, cardiac, muscle, and spinal
tissue). As explained previously, the MEA-wells are organized in an
ANSI-SLAS compliant format, compatible with traditional plate
readers and automated instrumentation. Within each well, a
plurality (e.g., between 4 and 64 individual embedded
microelectrodes (.about.30-50 .mu.m diameter; .about.200-350 .mu.m
center-to-center spacing, in accordance with an exemplary
embodiment) with integrated ground electrodes are capable of
simultaneously monitoring the activity of individual cells. The
arrangement of these electrodes into a grid extends the recording
range up to a 1.5.times.1.5 mm area, providing concurrent access to
both single-cell and network-level activity.
[0049] FIGS. 7B and 7C provide more detailed views (i.e., zoom)
views of each electrode set associated with an embodiment of the
multiwell MEA 300 consistent with the present disclosure. According
to one embodiment, each electrode is constructed of nano-textured
gold on an FR4 epoxy resin with an optional reflective white
overlay to reflect light back into the culture well for increased
irradiance of the cell cultures. Nano-texturing of gold is achieved
through proprietary processes designed to increase the surface area
of gold, thereby lowering the electrode impedance and noise.
[0050] Each of the electrodes in every set of one or more
electrodes may be configured to simultaneously provide stimulation
to the culture well and record electric signal resulting from
stimulation of the cells. Alternatively or additionally, some of
the electrodes in the set may be configured to provide stimulation
only, while other electrodes are dedicated to recording cellular
activity.
[0051] As explained, processes and methods consistent with the
disclosed embodiments provide solutions for integrating optical
stimulation in a multiwell MEA 300. In addition to the structural
and functional aspect of the multiwell MEA 300, the present
disclosure is directed to processes and methods for using the
multiwell MEA 300 to perform high-throughput, large scale testing,
such as testing based on optogenetics techniques. Consequently, the
presently disclosed electro-optical stimulation delivery system 100
includes a computer system (or stimulation GUI) that has been
customized to control the stimulation of the culture wells and
provide an interface for collecting/analyzing the cellular activity
resulting from the stimulation.
[0052] FIG. 8 illustrates an exemplary schematic diagram associated
with the control and monitoring system 800 that is adapted to
interface with a multiwell microelectrode array (MEA) with optical
stimulation capabilities. As explained, control and monitoring
system 800 may include processor-based device that includes its own
microcontroller, volatile and non-volatile memory, one or more
databases, and one or more interfaces for communicating data with a
user.
[0053] According to one embodiment, control and monitoring system
800 may include one or more hardware components including, for
example, a central processing unit (CPU) or microprocessor 811, a
random access memory (RAM) module 812, a read-only memory (ROM)
module 813, a memory or data storage module 814, a database 815,
one or more input/output (I/O) devices 816, and an interface 817.
Alternatively and/or additionally, control and monitoring system
800 may include one or more software media components such as, for
example, a computer-readable medium including computer-executable
instructions for performing methods consistent with certain
disclosed embodiments. It is contemplated that one or more of the
hardware components listed above may be implemented using software.
For example, storage 814 may include a software partition
associated with one or more other hardware components of control
and monitoring system 800. Control and monitoring system 800 may
include additional, fewer, and/or different components than those
listed above. It is understood that the components listed above are
exemplary only and not intended to be limiting.
[0054] CPU 811 may include one or more processors, each configured
to execute instructions and process data to perform one or more
functions associated with control and monitoring system 800. As
illustrated in FIG. 8, CPU 811 may be communicatively coupled to
RAM 812, ROM 813, storage 814, database 815, I/O devices 816, and
interface 817. CPU 811 may be configured to execute sequences of
computer program instructions to perform various processes, which
will be described in detail below. The computer program
instructions may be loaded into RANI 812 for execution by CPU
811.
[0055] RAM 812 and ROM 813 may each include one or more devices for
storing information associated with an operation of control and
monitoring system 800 and/or CPU 811. For example, ROM 813 may
include a memory device configured to access and store information
associated with control and monitoring system 800, including, for
example, stimulation schemes for different types of experiments.
RAM 812 may include a memory device for storing data associated
with one or more operations of CPU 811. For example, ROM 303 may
load instructions into RAM 302 for execution by CPU 811.
[0056] Storage 814 may include any type of mass storage device
configured to store information that CPU 811 may need to perform
processes consistent with the disclosed embodiments. For example,
storage 814 may include one or more magnetic and/or optical disk
devices, such as hard drives, CD-ROMs, DVD-ROMs, or any other type
of mass media device. Alternatively or additionally, storage 814
may include flash memory mass media storage or other
semiconductor-based storage medium. Database 815 may include one or
more software and/or hardware components that cooperate to store,
organize, sort, filter, and/or arrange data used by control and
monitoring system 800 and/or CPU 811.
[0057] I/O devices 816 may include one or more components
configured to communicate information with a component or user
associated with control and monitoring system 800. For example, I/O
devices 816 may include a console with an integrated keyboard and
mouse to allow a user to input parameters associated with control
and monitoring system 800. I/O devices 816 may also include a
display including a graphical user interface (GUI) for providing a
network management console for network administrators to configure
control and monitoring system 800. I/O devices 816 may also include
peripheral devices such as, for example, a printer for printing
information associated with control and monitoring system 800, a
user-accessible disk drive (e.g., a USB port, a floppy, CD-ROM, or
DVD-ROM drive, etc.) to allow a user to input data stored on a
portable media device, a microphone, a speaker system, or any other
suitable type of interface device. I/O devices may be configured to
output network analysis results and traffic characteristics.
[0058] Interface 817 may include one or more components configured
to transmit and receive data via a communication network, such as
the Internet, a local area network, a workstation peer-to-peer
network, a direct link network, a wireless network, or any other
suitable communication platform. For example, interface 817 may
include one or more modulators, demodulators, multiplexers,
demultiplexers, network communication devices, wireless devices,
antennas, modems, and any other type of device configured to enable
data communication via a communication network. According to one
embodiment, interface 817 may be coupled to or include wireless
communication devices, such as a module or modules configured to
transmit information wirelessly using Wi-Fi or Bluetooth wireless
protocols.
[0059] FIGS. 9A, 9B, 9C, 9D, and 9E provide exemplary user
interface screens shots associated with control and monitoring
system 800. According to one embodiment, the software may be
Axion's Integrated Studio (AxIS) suite of software provided by
Axion BioSystems of Atlanta, Ga. This software may provide an
interface that allows users to create sophisticated stimulation
experiments via I/O devices 816 and/or interface 817. This software
allows concurrent monitoring of channel recordings, digital and
analog filter adjustments, electrode assignment, and stimulus
waveform design, all within the same application in a modular
layout.
[0060] Software associated with stimulation GUI provides an
interface that allows users to control the stimulation parameters
associated with the electrodes and light-emitting elements. FIG. 9A
illustrates an exemplary software interface for independently
controlling the stimulation parameters for each culture well. The
software interface may also allow the user to establish and control
stimulation parameters 900 associated with different groups of
target culture wells 910. According to one embodiment, the user can
control the current level and waveform type associated with
electrical stimulation. Alternatively or additionally, the software
interface may allow a user to control the optical stimulation
parameters, such as frequency and amplitude of optical
radiation.
[0061] Software associated with stimulation GUI may provide an
interface for monitoring real-time experiment data. FIG. 9B
illustrates a screen shot of a software recording module that
displays a real-time, or post-experiment, scrolling "raster" plot
display of action potentials detected by each electrode in a sample
MEA well. The timing of each detected action potential signal is
demarcated by a vertical line 920 for each electrode in a sample
MEA well. A depiction of delivered optical stimulation patterns of
one 930 or more wavelengths 940 is shown on the same display. This
conceptual example illustrates one way that delivered light
stimulation might be visualized, and furthermore visualized in a
way that is temporally aligned with electrical recordings and/or
electrical stimulation from the MEA electrodes.
[0062] FIG. 9C provides an exemplary, conceptual software
visualization illustrating metrics of detected signals that are
further classified and visualized as light-evoked. In this example,
beating from a cardiac cell culture is detected and analyzed, and
the timing between consecutive beats, termed the beat period, is
calculated and displayed as real-time beats 950. If the beats are
classified as being evoked by delivered light, they are represented
differently, as illustrated by beats 960, to allow the experimenter
to visually assess the effects of optical stimulation.
[0063] FIG. 9D provides another exemplary conceptual software
visualization, whereby an activity metric is calculated and
displayed in relation to delivered optical stimulation. In this
example, a peristimulus time histogram of detected action
potentials in relation to delivered pulses of light is generated
and continually updated. Indication 980 indicates the timing of
light delivery and the histogram 970 includes action potential
counts falling within bins of elapsed time after each delivered
light pulse.
[0064] FIG. 9E provides another exemplary conceptual software
visualization, whereby electrical activity 990 detected by each
recording electrode is displayed in real-time or after an
experiment. Characteristics of delivered light patterns, including
the timing, intensity, or wavelength 995, is/are indicated
graphically and superimposed on the plots of detected activity. The
optical stimulation patterns are displayed in a manner that is time
synchronized with the visualization of detected electrical
activity. Such representations allow the experimenter to easily
visually assess the impact of optical stimulation on detected
signals.
[0065] FIG. 10 illustrates an exemplary interface system for
communicatively coupling the presently disclosed multiwell
microelectrode array (MEA) with optical stimulation capabilities
with a processor-based computing system having an integrated data
analysis and control suite. An optical stimulation module 1000 is
shown here as a detachable platform that can be placed onto the
multiwell MEA plate 1010 while the plate is docked in the interface
system 1020.
[0066] FIG. 11 provides a schematic block diagram of certain
components associated with the exemplary interface system
illustrated in FIG. 10 and its corresponding interactions between
the multiwell MEA 300 and control and monitoring system 800. Such
an interface system may provide full stimulation and recording
access to a plate with 768 electrodes. Three banks of 64-channel
ICs interface the electrodes and multiplex the resulting signals by
a factor of 8. Additional multiplexing is used to interface to a
bank of ADCs, with FPGAs providing data communication, system
coordination, and data processing functions.
[0067] FIG. 12 provides a functional block diagram associated with
an exemplary process for use and control of the presently disclosed
multiwell microelectrode array (MEA) with optical stimulation
capabilities. Optical patterns 1210 are delivered to one or more
MEA wells. Subsequently or simultaneously, electrical signals are
detected 1220 by one or more electrodes in one or more MEA wells.
These detected signals are analyzed 1230 and output 1240 by the
system. Optionally, the illumination pattern may be adjusted 1250,
either by the experimenter or automatically by the optical
stimulation module or processor, based on the analysis of detected
signals 1230. For example, the intensity of delivered light pulses
might be automatically adjusted on a per-well basis to an optimal
level 1260, based on detected metrics of neural or cardiac cell
culture activity. This feedback-driven tuning of delivered light
might allow for advanced, algorithmic control over the state of
cell cultures on a per well basis, allowing for advanced control
and analysis of target cells and cell networks.
[0068] FIG. 13 provides a conceptual, perspective view of a
sectioned MEA well within a multiwell MEA plate 300. In this
embodiment, a transparent MEA plate lid 1330 remains on the MEA
plate during use. A light delivery platform, including an array of
LEDs 1310 on an electronics board 1300 substrate, and an array of
reflectors 1320 or lenses fits onto the multiwell plate. Recesses
in the plate lid allow for mechanical alignment, reduce the escape
of light into adjacent wells, and improve the optics of light
delivery. Light passes through a volume of cell culture medium 1350
which might optionally be transparent in color or colored such that
the delivered light is not absorbed by the medium. Lid, reflector,
and MEA plate modifications may be incorporated for the purpose of
maximizing efficiency and/or providing uniform light delivery to
the central target cell culture region 1360 above the grid of
microelectrodes.
[0069] FIG. 14A provides alternate configurations for the lid 1400,
reflector 1440, and LEDs 1430, shown in FIG. 13. In FIG. 14A, the
lid 1400 is recessed to allow mechanical fitting of the LED
reflector, to prevent light escape from the well, and to enable the
LEDs and reflector to be moved closer to the target tissue or cells
at the bottom of the MEA well. The lid is furthermore shaped with a
lens to refract light towards the center of the well, enhancing
light delivery to the sample. In 14B the lid is shaped into a
Fresnel lens, to provide similar refraction of light, while not
extending as far into the well. In 14C the lid is recessed to a
greater extent and the LEDs or LED reflector to extend more deeply
into the well, offering the potential for improved light delivery
or light delivery concentrated towards a region of the target
tissue.
[0070] It will be apparent to those skilled in the art that various
modifications and variations can be made to the disclosed multiwell
microelectrode arrays with integrated optical stimulation
capabilities and associated methods for using the same. Other
embodiments of the present disclosure will be apparent to those
skilled in the art from consideration of the specification and
practice of the present disclosure. It is intended that the
specification and examples be considered as exemplary only, with a
true scope of the present disclosure being indicated by the
following claims and their equivalents.
* * * * *