Note: Descriptions are shown in the official language in which they were submitted.
MULTI-WELL PLATES AND METHODS OF USE THEREOF
BACKGROUND
Plates containing a multiplicity of wells for holding samples of chemicals,
cells or other
biological materials for observation, are known in the art. Commonly, such
plates have a 3:2
aspect ratio and thus contain 24 (4 x 6), 96 (8 x12), 384 (16 x 24), or 1536
(32 x 48) wells; a
typical 96-well plate is 128 mm long and 86 mm wide, and standards for the
footprint and
bottom outside flange of 96-well plates are described in ANSI/SBS 1-2004 and
ANSI/SBS 3-
2004, respectively.
Such multi-well plates, also sometimes referred to as microwell plates or
microtiter
plates depending on the volume of the wells, are generally constructed of
plastic, e.g.
polystyrene, polypropylene or polycarbonate, or a combination of such
materials, in some cases
also incorporating glass in the bottom portion of the plate. In many
applications, the bottom
of the well is transparent to a frequency of light that will be used to
observe the sample. The
size of wells in terms of depth, height, and total volume, as well the shape
of the wells and the
shape of the bottoms of the wells, varies in accordance with the particular
use to which the
plate is to be put.
Examples of commercial suppliers of such plates are:
Perkin-Elmer (see
http://www.perkinelmer. com/CMSRe sources/Images/44 -
73879SPC_M icrop lateDimensionsSummary Chart.pdf);
Sigma-Aldrich (see http
://www.sigmaaldrich. co m/labware/labware -
products. html?TablePage=9576216); and
Thermo-Scientific (see
http://www.thermoscientific.com/ecomm/servlet/productscatalog_l 1152 81996_-
1_4).
One area in which widespread use of such plates is made is high-throughput
screening
for the testing of compounds in drug development, binding assays for antigens
and the like.
Often, in high-throughput screening and other applications, automated
machinery is
used to dispense a volume of liquid into some or all of the wells
simultaneously, for example,
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by dispensing fluid simultaneously into the wells of an 8-well row in a 96-
well plate or into
all the wells of a 384-well plate. However, if the amount of liquid added to a
specific well is
incorrect, such fact may not become known until the entire experiment is
completed; and if
the specific well to which the incorrect amount of fluid was added is not
identified, it may be
.. necessary to disregard the results for the entire plate.
BRIEF DESCRIPTION
There is provided in accordance with an embodiment of the invention a plate,
comprising a first substantially planar surface having at least one first
aperture defined
therein; a second substantially planar surface substantially parallel to the
first substantially
planar surface, the second substantially planar surface being spaced from the
first
substantially planar surface; at least one well defined within the plate, the
at least one well
having a second aperture corresponding to and in alignment with one of the at
least one first
aperture, the at least one well having a sidewall and a bottom and the at
least one well
extending from the first substantially planar surface toward the second
substantially planar
surface, the at least one well being displaceable away from the first
substantially planar
surface; and at least one signal provider, functionally associated with the at
least one well,
capable of producing a signal in response to displacement of the at least one
well away from
the first surface.
In some embodiments, the first surface has a plurality of first apertures
defined
therewithin, and a plurality of wells are defined within the plate, each well
having a second
aperture corresponding to and in alignment with a first aperture of the
plurality of first
apertures defined in the first surface, and each of the wells having a
sidewall and a bottom
and each well extending from the first substantially planar surface toward the
second
substantially planar surface. In some embodiments, each of the plurality of
wells is
displaceable away from the first substantially planar surface.
There is also provided in accordance with an embodiment of the invention a
multiwell
plate, comprising a first substantially planar surface having a plurality of
first apertures
defined therein; a second substantially planar surface substantially parallel
to the first
substantially planar surface, the second substantially planar surface being
spaced from the
first substantially planar surface; a plurality of wells defined within the
plate, each well
having a second aperture corresponding to and in alignment with one of the
first apertures
defined in the first surface, each of the wells having a sidewall and a bottom
and each well
extending from the first substantially planar surface toward the second
substantially planar
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surface, each of the wells being displaceable away from the first
substantially planar surface;
and at least one signal provider, functionally associated with the plurality
of wells, capable of
producing a signal in response to displacement of at least one the well away
from the first
surface.
In some embodiments, the first and second surfaces are spaced apart by a
plurality of
sidewalls extending between the first and second surfaces.
In some embodiments, movement of all the wells is coupled, so that the signal
provider is capable of providing a single signal in response to displacement
of any one or
more of the wells. In some embodiments, movement of some of the wells is
coupled into two
or more groups, and the at least one signal provider comprises multiple signal
providers, each
capable of providing a signal in response to displacement of one of the
groups. In some
embodiments, movement of some of the wells is coupled into two or more groups,
and the
signal provider is capable of providing a separate signal in response to
displacement of each
one of the groups.
In some embodiments, the at least one signal provider comprises a plurality of
signal
providers, each associated with one well of the plurality of wells, each of
the plurality of
wells is independently displaceable away from the first surface, and each of
the plurality of
signal providers is capable of providing a signal in response to displacement
of one of the
plurality of wells associated therewith.
In some embodiments, the at least one signal provider is capable of providing
a signal
in response to placement in a the well of 300 milligrams of material. 250
milligrams of
material, 200 milligrams of material, 150 milligrams of material, 100
milligrams of material,
75 milligrams of material. 50 milligrams of material, 45 milligrams of
material, 40
milligrams of material, 35 milligrams of material, 30 milligrams of material,
25 milligrams of
material, 20 milligrams of material, 15 milligrams of material, 10 milligrams
of material. 5
milligrams of material, 4 milligrams of material, 3 milligrams of material, 2
milligrams of
material, or 1 milligram of material, 500 micrograms ( g) of material, 300 g
of material,
200 g of material, or 100 lmg of material.
In some embodiments, the at least one signal provider is capable of providing
a signal
in response to placement in a the well of 300 microliters (pi) of fluid, 250
I of fluid, 200 1
of fluid, 150 I of fluid, 100 I of fluid, 75 I of fluid. 50 I of fluid, 45
I of fluid. 40 I of
fluid, 35 1 of fluid, 30 1 of fluid, 25 1 of fluid, 20 I of fluid, 15 1
of fluid, 10 1 of fluid,
5 I of fluid, 4 1 of fluid, 3 I of fluid, 2 1 of fluid, 1 1 of fluid, 0.5
I of fluid, 0.3 1 of
fluid, 0.5 I of fluid, or 0.1 I of fluid.
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In some embodiments, the signal provider is capable of providing a signal in
response
to displacement of at least one the well toward the first surface.
In some embodiments, the at least one signal provider is capable of providing
a signal
in response to removal from a the well of 300 milligrams of material, 250
milligrams of
material, 200 milligrams of material, 150 milligrams of material, 100
milligrams of material,
75 milligrams of material. 50 milligrams of material, 45 milligrams of
material, 40
milligrams of material, 35 milligrams of material, 30 milligrams of material,
25 milligrams of
material, 20 milligrams of material, 15 milligrams of material, 10 milligrams
of material. 5
milligrams of material, 4 milligrams of material, 3 milligrams of material, 2
milligrams of
material, or 1 milligram of material, 500 micrograms ( g) of material, 300 g
of material,
200 g of material, or 100 g of material.
In some embodiments, the at least one signal provider is capable of providing
a signal
in response to removal from a the well of 300 microliters ( 1) of fluid, 250
1 of fluid. 200 1
of fluid, 150 1 of fluid, 100 .1 of fluid, 75 1 of fluid, 50 1 of fluid,
45 1 of fluid, 40 1 of
fluid, 35 I of fluid, 30 1 of fluid. 25 1,11 of fluid, 20 I of fluid, 15
ittl of fluid, 10 1 of fluid,
5 1 of fluid, 4 1 of fluid, 3 1 of fluid, 2 1 of fluid, 1 1 of fluid, 0.5
1 of fluid, 0.3 1 of
fluid, 0.2 I of fluid, or 0.1 I of fluid.
In some embodiments, at least one well in the plate is removable therefrom.
In some embodiments, the plate comprises at least one temperature sensor
associated
with at least one of the wells. In some embodiments, the temperature sensor is
located in or
on one of the wells. In some embodiments, the at least one temperature sensor
is
configured to provide a signal representing a temperature in the at least one
well or in a
vicinity thereof. In some such embodiments, the at least one temperature
sensor is configured
to continuously detect the temperature in the at least one well, and to
periodically provide the
signal representing the temperature.
In some embodiments, the plate further comprises an electronic storage element
for
storage of at least one signal provided by at least one of the at least one
signal provider and
the at least one temperature sensor.
In some embodiments, the at least one temperature sensor is configured to
detect the
temperature in a group of wells from the plurality of the wells.
In some embodiments, the at least one temperature sensor comprises a single
temperature sensor configured to detect the temperature in all the wells.
4
In some embodiments each signal provider of the at least one signal provider
comprises:
at least one flexible arm attached at a first end thereof to a support and at
a second end
thereof engages at least a portion of said at least one well; and
at least one strain gauge disposed on said at least one flexible arm and
adapted to detect
deflection of said at least one flexible arm.
In some embodiments each signal provider of the at least one signal provider
further comprises
at least one electronic card, functionally associated with said at least one
strain gauge, and
adapted to correlate the detected deflection of said at least one flexible arm
with a change in
content of said at least one well causing said displacement of said at least
one well away from
said first surface.
In some embodiments the signal provider includes a first flexible arm having
mounted thereon a
first strain gauge functionally associated with a first electronic card, and a
second flexible arm
having mounted thereon a second strain gauge functionally associated with a
second electronic
card, said second flexible arm being disposed in parallel to said first
flexible arm.
In accordance with embodiments of the present invention first and second
substantially planar
surfaces are spaced apart by a plurality of sidewalls extending between the
first and second
substantially planar surfaces.
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In some embodiments, the at least one temperature sensor comprises a plurality
of
temperature sensors, each associated with one of the plurality of wells for
detecting the
temperature in the one of the plurality of wells associated therewith.
In some embodiments, the plate further comprises at least one heating
component
associated with the at least one well, the at least one heating component
being located in
sufficient proximity to the at least one well to heat the at least one well or
its interior. In some
embodiments, the at least one heating component comprises a plurality of
heating
components, each associated with one well of the plurality of wells and
located in sufficient
proximity to the one well associated therewith to heat the one well or its
interior, without
substantially heating others of the plurality of wells. In some embodiments,
the at least one
heating component comprises a heating coil. In some embodiments, the at least
one heating
component is also capable of cooling the at least one well. In some
embodiments, the heating
component comprises a Peltier device.
In some embodiments, at least one well in the plate is removable therefrom, in
a
manner that does not remove from the plate a heating component associated with
the at least
one removable well. In some embodiments, at least one well in the plate is
removable
therefrom, and the heating component associated with the at least one
removable well is
attached to or formed integrally with the at least one well and is removable
therewith.
In some embodiments, the plate further comprises an electrical port
functionally
associated with at least one of the at least one signal provider and the at
least one temperature
sensor. In some embodiments, the plate further comprises a rechargeable power
supply,
functionally associated with at least one of the at least one signal provider
and the at least one
temperature sensor, and configured to be recharged by connection thereof to a
power source.
In some embodiments, the rechargeable power supply is configured to be
recharged when the
electrical port is electrically connected to a power source.
There is also provided in accordance with an embodiment of the invention a
data
reader configured to receive therein a plate according embodiments of the
present invention,
the data reader comprising a base for placement of the plate thereon; an
electrical port
corresponding to the electrical port of the plate for electrical engagement
therewith; and a
processor functionally associated with the electrical port, for processing
signals obtained
from at least one of the signal provider and the temperature sensor via the
electrical port. In
some embodiments, the processor is configured to obtain the signals directly
from at least one
of the signal provider and the temperature sensor via the electrical port. In
some
embodiments, the processor is configured to obtain the signals from an
electronic storage
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component storing at least one signal provided by at least one of the signal
provider and the
temperature sensor.
In some embodiments the data reader further comprises a display functionally
associated with the processor, the display configured to provide to a user
information
obtained from the processed signals. In some embodiments, the information
comprises an
indication of at least one of: (i) an amount of fluid in the plate at a
specific time; (ii) an
amount of fluid in at least one the well at a specific time; (iii) a change in
an amount of fluid
in the plate over a period of time; (iv) a change in an amount of fluid in at
least one the well
over a period of time; (v) a temperature of at least one the well at a
specific time; and (vi) a
change in temperature of at least one the well over a period of time. In some
embodiments,
the processor is configured to process the signals in real time and the
display is configured to
provide to the user the information in real-time.
There is also provided in accordance with an embodiment of the invention a
plate
comprising a first substantially planar surface having at least one first
aperture defined
therein; a second substantially planar surface substantially parallel to the
first substantially
planar surface, the second substantially planar surface being spaced from the
first
substantially planar surface; at least one well defined within the plate, the
at least one well
having a second aperture corresponding to and in alignment with one of the at
least one first
apertures defined in the first surface, the at least one well having a
sidewall and a bottom and
the at least one well extending from the first substantially planar surface
toward the second
substantially planar surface; and at least one heating component associated
with the at least
one well, which is located in sufficient proximity to the at least one well to
heat the at least
one well or its interior. In some embodiments, the first surface has a
plurality of first
apertures defined therewithin; the at least one well comprises a plurality of
wells defined
within the plate, each well having a second aperture corresponding to and in
alignment with
one first aperture from the plurality of first apertures defined in the first
surface, each of the
wells having a sidewall and a bottom and each well extending from the first
substantially
planar surface toward the second substantially planar surface; and the at
least one heating
component comprising a plurality of heating components such that each of the
wells has one
of the plurality of heating components associated therewith, each of the
heating components
being located in sufficient proximity to the well with which the heating
component is
associated to heat the well or its interior without substantially heating
other wells. In some
embodiments, the heating component comprises a heating coil. In some
embodiments, the
heating component is also capable of cooling the well. In some embodiments,
the heating
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component comprises a Peltier device. In some embodiments, at least one well
in the plate is
removable therefrom, without removing from the plate a heating component
associated with
the at least one removable well. In some embodiments, at least one well in the
plate is
removable therefrom, and the heating component associated with the at least
one removable
well is attached to or formed integrally with the at least one well and is
removable therewith.
There is also provided in accordance with an embodiment of the invention a
method
for measuring the amount of fluid added to a plate according to any one of the
embodiments
of the present invention, the method comprising recording an initial signal
provided by the
signal provider, and after a fluid has been added to at least one well in the
plate, obtaining a
second signal generated by the signal provider in response to the addition of
the fluid,
wherein, on the basis of a difference between the initial signal and the
second signal, the
amount of the fluid added to the at least one well can be calculated. In some
embodiments,
the method further comprises adding fluid to the plate after the recording an
initial signal and
before the obtaining a second signal. In some embodiments, the method further
comprises on
the basis of the difference between the initial signal and the second signal,
calculating an
amount of the fluid added to the at least one well. In some embodiments, the
calculating an
amount comprises calculating a volume of the fluid added to the at least one
well. In some
embodiments, the calculating an amount comprises calculating a mass of the
fluid added to
the at least one well. In some embodiments, the calculating an amount
comprises calculating
a volume and a mass of the fluid added to the at least one well.
There is also provided in accordance with an embodiment of the invention a
method
for measuring the amount of fluid lost from a plate according to any one of
the embodiments
of the present invention, the plate having an initial amount of fluid disposed
in at least one
well of the plate, the method comprising recording an initial signal provided
by the signal
provider at a first time; obtaining from the signal provider a second signal
at a second time
after the first time; and on the basis of the difference between the initial
signal and the second
signal, calculating an amount of the fluid lost from the at least one well of
the plate. In some
embodiments, the calculating an amount comprises calculating a volume of the
fluid lost
from the at least one well. In some embodiments, the calculating an amount
comprises
calculating a mass of the fluid lost from the at least one well. In some
embodiments, the
calculating an amount comprises calculating a volume and a mass of the fluid
lost from the at
least one well. In some embodiments, the method further comprises periodically
repeating the
step of obtaining a signal, and on the basis of the difference between signals
obtained at two
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different times, calculating an amount of fluid lost from the at least one
well in a duration
between the two different times.
There is also provided in accordance with an embodiment of the invention a
method
comprising obtaining a baseline measurement of displacement of at least one
well in a multi-
well plate having disposed therein a displacement measuring assembly for
measuring the
displacement of at least one well in the plate in response a change in an
amount of fluid in the
at least one well, the baseline measurement being obtained via the
displacement measuring
assembly; at a time after the obtaining of the baseline measurement, obtaining
a second
measurement of displacement of the at least one well; and on the basis of the
second
measurement of displacement, calculating the change in the amount of fluid in
the at least one
well.
There is also provide in accordance with an embodiment of the invention a
method
comprising at a first time, obtaining a baseline measurement of displacement
of at least one
well in a multi-well plate having disposed therein a displacement measuring
assembly for
measuring the displacement of at least one well in the plate in response a
change in an
amount of fluid in the at least one well, the baseline measurement being
obtained via the
displacement measuring assembly; at a second time after the first time,
measuring the
displacement of the at least one well; and on the basis of a change in
displacement between
the first and second times, calculating a change in the amount of fluid in the
at least one well,
wherein at at least one of the first and second times, a detectable amount of
fluid is present in
the well. In some embodiments, the method further comprises periodically
repeating the step
of measuring the displacement of the at least one well at a second time, and
on the basis of a
change in displacement between two the measurements of displacement,
calculating a change
in the amount of fluid in the at least one well during a period between the
two the
measurements of displacement. In some embodiments, the change in the amount of
fluid is
due to addition of fluid to the at least one well. In some embodiments, the
change in the
amount of fluid is due to loss of fluid from the at least one well.
In some embodiments of the aforemention methods, the plate is a plate
according to
any one of the embodiments of the present invention.
In some embodiments of the aforemention methods, the signal provider is
sufficiently
sensitive to detect a change of 300 microliters (IA). 250 I, 200 I, 150 I,
100 I, 75 I, 50
IL, 45 IL, 40 IL, 35 1, 30 1. 25 1, 20 1, 15 1, 10 1, 5 1, 4 1, 3
Ill, 2 I, 1 IL, 0.5 1 of
fluid, 0.3 1 of fluid. 0.2 1 of fluid, or 0.1 1 of fluid, in the volume of
fluid in the at least
one well.
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In some embodiments of the aforemention methods, the signal provider is
sufficiently
sensitive to detect a change of 300 milligrams (mg), 250 mg. 200 mg, 150 mg.
100 mg, 75
mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 4 mg,
3 mg, 2
mg, 1 mg. 500 micrograms (pig), 300 vg, 200 lag, or 100 pg in the mass of
fluid in the at least
one well.
In some embodiments of the aforemention methods, the method further comprises
detecting a temperature in at least one well. In some embodiments, the method
further
comprises detecting the temperature in the at least one well at at least two
different points in
time. In some embodiments, the method further comprises adjusting the
temperature of an
individual well in response to the detecting the temperature.
In some embodiments of the aforemention methods, at least one well in the
multi-well
plate is removable therefrom.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
the invention
pertains. In case of conflict, the specification, including definitions, will
take precedence.
As used herein, the terms "comprising", "including", "having" and grammatical
variants thereof are to be taken as specifying the stated features, integers,
steps or
components but do not preclude the addition of one or more additional
features, integers,
steps, components or groups thereof. These terms encompass the terms
"consisting of" and
"consisting essentially of".
As used herein, the indefinite articles "a" and "an" mean "at least one" or
"one or
more" unless the context clearly dictates otherwise.
Embodiments of methods and/or devices of the invention may involve performing
or
completing selected tasks manually, automatically, or a combination thereof.
Some
embodiments of the invention are implemented with the use of components that
comprise
hardware, software, firmware or combinations thereof. In some embodiments,
some
components are general-purpose components such as general purpose computers or
monitors.
In some embodiments, some components are dedicated or custom components such
as
circuits, integrated circuits or software.
For example, in some embodiments, some of an embodiment is implemented as a
plurality of software instructions executed by a data processor, for example
which is part of a
general-purpose or custom computer. In some embodiments, the data processor or
computer
comprises volatile memory for storing instructions and/or data and/or a non-
volatile storage,
for example, a magnetic hard-disk and/or removable media, for storing
instructions and/or
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data. In some embodiments, implementation includes a network connection. In
some
embodiments, implementation includes a user interface, generally comprising
one or more of
input devices (e.g., allowing input of commands and/or parameters) and output
devices (e.g.,
allowing reporting parameters of operation and results.
BRIEF DESCRIPTION OF THE FIGURES
Some embodiments of the invention are described herein with reference to the
accompanying figures. The description, together with the figures, makes
apparent to a person
having ordinary skill in the art how some embodiments of the invention may be
practiced.
The figures are for the purpose of illustrative discussion and no attempt is
made to show
structural details of an embodiment in more detail than is necessary for a
fundamental
understanding of the invention. For the sake of clarity, some objects depicted
in the figures
may not be to scale.
In the Figures:
Fig. 1 is a perspective view of a multi-well plate of the prior art;
Fig. 2A is a perspective view of a multi-well plate constructed and operative
in
accordance with an embodiment of the invention;
Fig. 2B is an exploded view of the multi-well plate of Fig. 2A;
Figs. 2C and 2D are perspective sectional views of the multi-well plate of
Figs. 2A
and 2B, taken along section lines IIC-IIC and IID-IID in Fig. 2A,
respectively;
Figs. 3A and 3B are perspective views of a multi-well plate constructed and
operative
in accordance with another embodiment of the invention;
Fig. 3C is an exploded view of the multi-well plate of Figs. 3A and 3B;
Fig. 3D is an enlarged perspective view of supports, arms, and blocks forming
part of
the multi-well plate of Figs. 3A and 3B;
Figs. 3E, 3F, and 3G are sectional views of the multi-well plate of Figs. 3A
to 3C,
taken along section lines IIIE-IIIE, IIIF-IIIF, and IIIG-IIIG in Fig. 3A;
Fig. 4A is a perspective view of a multi-well plate constructed and operative
in
accordance with yet another embodiment of the invention;
Fig. 4B is an exploded view of the multi-well plate of Fig. 4A;
Fig. 4C is a sectional view of the multi-well plate of Figs. 4A and 4B, taken
along
section lines IVC-IVC in Fig. 4A;
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Figs. 5A ¨ 5D are screen shots illustrating a graphical user interface for on-
line (real
time) monitoring of addition of fluid to a multi-well plate in accordance with
embodiments of
the teachings herein;
Figs. 6A and 6B are screen shots illustrating a graphical user interface for
off-line
volume monitoring of fluid in a multi-well plate in accordance with
embodiments of the
teachings herein;
Fig. 7 is a screen shot illustrating a graphical user interface for off-line
temperature
monitoring of fluid in a multi-well plate in accordance with embodiments of
the invention;
Fig. 8 is a perspective view of a plate base and data reader constructed and
operative
in accordance with an embodiment of the teachings herein, for receiving
signals from a multi-
well plate in accordance with the teachings herein; and
Figs. 9A and 9B are perspective views of a device for removing well-containing
elements or well-defining elements from and/or for emplacing such elements in
a multi-well
plate, the device constructed and operative in accordance with an embodiment
of the
teachings herein.
DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION
The principles, uses and implementations of the teachings herein may be better
understood with reference to the accompanying description and figures. Upon
perusal of the
description and figures present herein, one skilled in the art is able to
implement the invention
without undue effort or experimentation.
Before explaining at least one embodiment of the invention in detail, it is to
be
understood that the invention is not limited in its applications to the
details of construction
and the arrangement of the components and/or methods set forth in the
following description
and/or illustrated in the drawings and/or the Examples. The invention can be
implemented
with other embodiments and can be practiced or carried out in various ways. It
is also
understood that the phraseology and terminology employed herein is for
descriptive purpose
and should not be regarded as limiting.
Reference is now made to Fig. 1, which is a perspective view of a multi-well
plate of
the prior art. Figure 1 shows a typical 96-well plate 110 as is known in the
art. Plate 10 has an
upper surface 12, a lower surface 14, and a plurality of sides 16 between the
upper and lower
surfaces 12 and 14. Extending between surfaces 12 and 14 are a plurality of
wells 17, ninety-
six such wells in all, arranged in eight rows of twelve wells each. Each well
has an aperture
18 formed in upper surface 12, to facilitate the injection of sample fluid
into the well. Plate
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may be made of plastic, such as polystyrene or polycarbonate, or a combination
of such
materials; in some cases, it may have a glass bottom. Typically the lower
surface 14 is
transparent at at least some particular frequency or range of frequencies of
light at which the
sample placed in the well will be observed, although for some uses such
transparency may
5 not be
necessary. Sides 20 of the wells 17 may or may not be transparent, depending
on the
nature of the sample and the type of observations to be made.
The wells are typically of circular cross-section in the x-y plane and of
essentially
cylindrical shape, but they may have cross-sections of other shapes, such as
rectangular or
square, and they may have different shapes along their lengths, for example
the sides of the
10 wells
may taper from the upper opening to the bottom of the well along a portion
thereof or
along their entire length. The bottoms of the wells are typically flat but,
depending upon the
intended use of the plate, may be formed with other shapes, such as conical,
frusto-conical, or
spherical bottoms (i.e. V- or U-shaped bottom cross sections when viewed along
the z-axis).
A plate such as plate 10 is typically of 85-86 mm width and 127-128 mm length,
with
an overall height that varies within the range of 10-20 mm, and with the
centers of the wells
being spaced 9 mm apart along the x and y axes. Standards for the footprint
and bottom
outside flange of 96-well microplates are described in ANSFSBS 1-2004 and
ANSI/SBS 3-
2004, respectively. That said, the plate may have different length, width, and
height
dimensions, may have a different distance between the wells, and may have a
different
number of wells, as is suitable for the specific application or use of the
plate. For example,
the plate may be a 384-well plate including 384 wells, arranged in sixteen
rows of twenty
four wells each, such that the centers of the wells are 4.5 mm apart along the
x and y axes.
Standards for the footprint and bottom outside flange of 384-well microplates
are described
in ANSI/SBS 1-2004 and ANSI/SBS 3-2004, respectively.
Reference is now made to Fig. 2A, which is a perspective view of a multi-well
plate
100 constructed and operative in accordance with an embodiment of the
invention, to Fig.
2B, which is an exploded view of the multi-well plate 100 of Fig. 2A, and to
Figs. 2C and
2D, which are perspective sectional views of the multi-well plate 100 of Figs.
2A and 2B,
taken along section lines IIC-IIC and IID-IID in Fig. 2A, respectively.
As shown in Fig. 2A, plate 100 is designed for use with existing equipment and
is
therefore sized in accordance with standard plate sizes currently in use, with
its wells
similarly spaced, as defined in ANSYSBS 1-2004 and in ANS1/SBS 3-2004. As
such, when
assembled, plate 100 looks similar to a typical 96-well plate, with an upper
surface 112, a
lower surface 114, and a plurality of sides 116 between surfaces 112 and 114.
A plurality of
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wells 117 are formed in plate 100 and extend between surfaces 112 and 114.
Each well has an
aperture 118 formed in upper surface 112, to facilitate the injection of
sample fluid into the
well.
Turning to Fig. 2B, which is an exploded view of plate 100, it is seen that
plate 100 is
actually formed of several parts. Sides 116 form part of a frame 122, which
has formed on an
inner portion thereof, at each lengthwise end of the frame, a pair of supports
124a and 124b,
which supports will be explained in more detail in connection with Figs. 2C
and 2D. Each of
the supports 124a and 124b has attached thereto a pair of flexible arms 126,
consisting of an
upper aim 126a and a lower arm 126b, wherein each arm is attached at the
proximal end
thereof to one of the supports, and is attached the distal end thereof to a
block 128. As seen in
the enlarged portion of Fig. 2B, each block 128 includes an upper portion and
a lower
portion, with the arms 126a and 126b being attached to lower surfaces 128a and
128b of the
upper and lower portions, respectively. The flexible arms 126 may be made of a
material,
generally metal or plastic, which is suitably flexible that it will be
sensitive to the addition of
a few micrograms weight to the wells, as will be explained in more detail
below. The arms
may be attached to blocks 128 by suitable means, such as adhesive or in some
cases melting
or welding.
Cylindrical side walls of wells 117 are formed in a well-containing element
130,
which may be formed of plastic, glass, or another suitable material. The well
containing
element 130 includes upper surface 112 and has flanges 132 at longitudinal
ends thereof.
When plate 100 is assembled, each flange 132 rests on, and optionally is
attached to, upper
surfaces of upper portions 128a of two blocks 128, one block at each end of
the flange, and in
some embodiments also on supports 124a and 124b (see Fig. 2D). A bottom piece
134, which
is a piece of plastic or glass approximately 170-1000 microns in thickness, is
sealingly
attached to the underside of well-containing element 130, so as to form the
bottom of each
well 117 in a manner that seals each well at that end.
In the embodiment shown in Figs. 2A-2D, the circumference of the uppermost
portion
of well-containing element 130, including the flanges 132, is slightly less
than the inner
circumference of frame 122. Consequently, when the flanges 132 of well-
containing element
130 rest on blocks 128, there is a small gap 138 between the well-containing
element 130 and
the frame 122 (see Fig. 2D). The presence of gap 138 allows for movement of
the well-
containing element 130 along the vertical axis and for concomitant deformation
of arms 126,
as will be explained below. In order to seal the gap 138 without inhibiting
such movement, a
very thin (approximately 7 micron) film 140 of a flexible material, such as a
suitable plastic,
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is attached to an upper edge 142 of the frame 122 and to an outer edge of
upper surface 112
of well-containing element 130. One way to accomplish such attachment to well-
containing
element 130 is by having a slightly raised rim 143 around the circumference of
the upper
edge of element 130, so that film 140 engages the rim 143 and, when also
attached to an outer
edge of upper surface 112, is disposed above gap 138.
As shown in Figs. 2A and 2B. film 140 is hollow in the center, so that most of
upper
surface 112, and particularly the area of surface 112 including wells 117, is
exposed and film
140 covers just the edge of well-containing element 130, gap 138, and upper
edge 142 of the
frame 122. However, it will also be appreciated that in some embodiments, film
140 may be
formed to cover the upper edge 142 of frame 122 as well as the entire upper
surface 112 of
element 130, in which case film 140 will be formed with a plurality of
circular openings
which would be aligned with apertures 118 in well-containing element 130, to
allow user
access to wells 117. In such embodiments, well-containing element 130 may be
formed with
slightly raised portions, or rims, around apertures 118, so as to provide
additional surfaces to
which film 140 may be affixed.
Film 140, well-containing element 130, bottom piece 134, and frame 122, are
attached
to each other using any suitable means, such as soldering, adhering, melting,
bonding, or any
other suitable attachment mechanism.
As noted, when plate 100 is assembled, well-containing element 130 rests on,
and is
optionally attached at each of its longitudinal ends to, a pair of blocks 128.
This restricts the
motion of element 130 in the x- and y-directions, but allows for motion in the
vertical (z)
direction. Thus, when fluid is placed into wells 117 in element 130, the
weight of the fluid
will result in downward displacement of element 130 and in deformation of arms
126. It will
be appreciated that typically not more than several hundred microliters of
fluid, and in some
applications only a few microliters of fluid, are added to a given well 117.
Consequently, the
flexible arms 126 should be of suitable flexibility to deflect in response to
the addition of
micrograms of fluid to the wells.
Reference is additionally made to Figs. 2C and 2D show in greater detail the
supports
124a and 124b, the arms 126 and blocks 128, and the attachment of the arms 126
to the
supports. Fig. 2C shows plate 100, as in Fig. 2A, but with a perspective cross-
sectional view
showing the end of the plate, the cross-sectional view taken along section
lines TIC-TIC in
Fig. 2A. Fig. 2D shows the same plate, with an enlarged cross-sectional view
taken from the
same angle as Fig. 2C along section lines 11D-11D in Figure 2A, which cross-
sectional view
shows the other end of plate. Fig. 2C shows the pair of arms 126 closest to
the end of the
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plate, whereas Fig. 2D shows the pair of aims 126 disposed slightly inward
from the end of
the plate.
Support 124a protrudes from both the inner wall 122a which is located at one
longitudinal end of frame 122, as well as from the longitudinal wall 122b
adjacent thereto.
Support 124b protrudes from the opposite longitudinal wall 122c and is spaced
from the inner
wall 122a by the width of support 124a. Supports 124a and 124b project in
opposite
directions from the longitudinal walls, each of supports 124a and 124b
projecting about one
third of the way between the longitudinal walls, with each support having a
pair of tongues
(shown in Fig. 2D for support 124b as 124b' and 124b") that project further
until
approximately midway between the two longitudinal walls. The distance between
the upper
faces of the tongues is equal to the distance between the arms 126a and 126b.
One pair of
arms is attached at its proximal end to support tongues 124a' and 124a" (not
shown) of
support 124a and the other pair of arms is attached at its proximal end to
tongues 124b' and
124b" of support 124b. Each pair of arms is attached at its distal end to a
block 128. As
noted, element 130 is placed so as to rest upon and be attached to blocks 128.
If this is
position is considered the neutral or ground position, arms 126 are
constructed so as to have
sufficient flexibility in the z-axis to deform from this neutral position upon
the addition of
liquid to some of wells 117.
Attached to the upper and lower surfaces of each arm 126, in the free region
of the
arm but, in some embodiments, near the point at which the arm is attached to
frame 122,
there is a thin, flat strain gauge 144. Each of the strain gauges is
electrically connected (e.g.
by thin wires, not shown) to a thin, flat electronic card 146 located below
the lower arms
126b. Preferably, electronic cards 146 are positioned so as to minimize the
length of the
connections between the strain gauges 144 and the cards 146. Cards 146 are
electrically
coupled to a processor (not shown). It will be appreciated that use of strain
gauges 144 and
cards 146 allows for correlation of the deflection of the arms 126 to the
change in electrical
resistance in a circuit, for example measured using a Wheatstone Bridge, which
is also
located on card 146, allowing calculation of the mass of the fluid added to
the wells. As such,
arms 126 together with strain gauges 144 form a signal provider, for providing
a signal
indicative of a change in the amount of fluid in one or more of the wells. If
the density of the
fluid is known, this facilitates computation of the volume of fluid added. In
some
embodiments, plate 100 further includes a power supply (not shown) such as a
rechargeable
battery, for example connected to electronic card 146, which power supply
provides power to
CA 02931068 2016-05-18
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electronic components of plate 100 and may be recharged when plate 100 is
connected to a
power source or computation device via a suitable port (not shown).
For example, if rows A through L of plate 100 are filled sequentially using an
8-tip
pipette, it is possible to iteratively calculate the amount of fluid added to
each row, and
therefore to identify in real time, on a row-by-row basis, when an incorrect
amount of fluid
has been added to the row. This allows that particular row to be discarded
from the
calculations at the end of the experiment, rather than discarding the results
for the entire
plate. Preferably, the apparatus used to add fluid to the wells will be
equipped with control
software to allow the affected row only to be excluded from further
manipulations during the
remainder of the experiment, such as the addition of reagents and reactants.
It will also be
appreciated that even if all the wells are filled simultaneously, the use of
plate 100 in
conjunction with appropriate software to identify in real time the addition of
an incorrect
amount of fluid to the plate enables the user to stop running experiments
using that particular
plate, thus avoiding waste of reagents, reactants and the like in downstream
experiments.
In accordance with some embodiments of the invention, plate 100 includes means
for
heating and, optionally, cooling individual wells. Such heating means may take
the form of,
for example. (a) a heating coil disposed around at least a portion of the
well. or (b) a Peltier
device, sometimes called a Peltier heat pump or thermoelectric cooler. As will
be appreciated,
a Peltier device can he used to cool as well as to heat an individual well, as
well as to sense or
monitor the temperature in the well or in the vicinity thereof. In this way,
the temperature in
individual wells may be controlled, for example the temperature in each well
may be
maintained at 37 C 0.5 C.
In one embodiment, a single heating coil or Peltier device may be disposed on
the
well-containing element 130 for collectively heating two or more wells 117. In
another
embodiment, heating coils or Peltier devices may be disposed on one or more
individual
wells 117 for heating thereof.
It will also be appreciated that if the temperature of the wells is
periodically
measured, and if the device used to measure the temperature is coupled to a
controller that
controls the heating means for each individual well, then the individual well
heating means,
used in conjunction with the periodic measuring of temperature in individual
wells, can
provide a way to improve control over the conditions in a given well. Thus,
for example,
plates 100 having such heating means may be stored in an incubator and the
temperature of
the wells monitored periodically and the temperature of individual wells
adjusted, if
necessary, by heating (or cooling) individual wells. Alternatively, the
heating means may
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themselves be used to effect incubation, for example temperature monitoring
and adjustment
may be effected frequently, say for example every 15, 10 or 5 minutes, to
maintain the
temperature in particular wells at e.g. 37 C 0.5 C so as to effect
incubation. Thus, by
equipping the plate 100, the well-containing element 130, or the wells 117
with individual
heating elements for each well, in cases in which it is found that the
temperature is incorrect,
the temperature may be adjusted and controlled.
It will be appreciated that since reagents and other fluids may be added into
or
removed from wells 117 in well-containing element 130 which may be removed
from plate
100 and optionally disposed of, plate 100 may be used multiple times and/or
for multiple
experiments, provided that well-containing element 130 are replaced between
each use of the
plate 100.
Reference is now made to Figs. 3A to 3G, which show a multi-well plate 300 and
components thereof, constructed and operative in accordance with embodiments
of the
invention. Specifically, Figs. 3A and 3B are perspective views of multi-well
plate 300, Fig.
3C is an exploded view of the multi-well plate 300. Fig. 3D is an enlarged
perspective view
of supports, arms, and blocks forming part of the multi-well plate 300, and
Figs. 3E, 3F, and
3G are sectional views of the multi-well plate 300 taken along section lines
IIIE-IIIE, IIIF-
IIIF, and IIIG-IIIG in Fig. 3A.
As shown in Figs. 3A and 3B, plate 300 is designed for use with existing
equipment
and is therefore sized in accordance with standard plate sizes currently in
use, with its wells
similarly spaced. Thus, when assembled, plate 300 looks similar to a typical
96-well plate,
with an upper surface 312, a lower surface 314, a plurality of sides 316
between surfaces 312
and 314, and having a plurality of wells 317 formed therein.
As shown in Fig. 3C, which is an exploded view of plate 300, plate 300 is
actually
formed of several parts. Sides 316 are part of a frame 322, which has formed
on an inner
portion thereof, at the lengthwise ends of the frame, a pair of supports 324a
and 324b each
including a plurality of "stairs" 325, which supports will be explained in
more detail in
connection with Figs. 3D, 3F, and 3G. Frame 322 includes end walls 322a and
322d disposed
at longitudinal ends of the frame, as well as longitudinal walls 322b and
322c.
Each of the supports 324a and 324h has attached thereto multiple flexible arms
326,
wherein each arm is attached at the proximal end thereof to one of the
supports, and is
attached the distal end thereof to a block 328. The structure and
functionality of flexible arms
326 and of blocks 328 will be described in further detail hereinbelow with
reference to Figs.
3D, 3F, and 3G. The flexible arms 326 may be made of a suitable material,
generally metal or
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plastic, which is suitably flexible as to be sensitive to the addition or
removal of a small
volume to or from the wells, as will be explained in more detail below. For
example, flexible
arms 326 would be sensitive to the addition to the wells of less than 300
microliters ( 1), less
than 250 I, less than 200 I, less than 150 1, less than 100 1, less than
75 I, less than 50
pi, less than 45 pi, less than 40 pl, less than 35 pl, less than 30 pl, less
than 25 pi, less than
20 1, less than 15 1, less than 10 1, less than 5 1, less than 4 1, less
than 3 1, less than 2
1, or less than 1 1.
The arms 326 may be attached to blocks 328 by suitable means, such as adhesive
or in
some cases melting or welding.
Plate 300 further includes eight identical well-supporting elements 330, each
including a longitudinal aperture 333 suited to receive a well-containing
element 348 therein.
Each well-containing element 348 has twelve cylinders 320, forming side walls
of tubular
wells 317, formed therein. Each of elements 330 also has a pair of flanges
332, one flange at
each of the longitudinal ends thereof. Well-supporting elements 330 may be
formed of plastic
or another suitable material and may contain other materials such as glass.
When assembled,
each flange 332 rests on, and preferably is attached to, two blocks 328, one
block per flange;
the manner in which blocks 328 are held in place relative to frame 322 will be
discussed in
more detail below.
A plurality of bottom pieces 334, which are pieces of plastic or glass
approximately
170-1000 microns in thickness, are sealingly attached to the undersides of
well-containing
elements 348, so as to form the bottom of each well 317 in a manner that seals
each well at
that end of the well. In the illustrated embodiment. three pieces 334, each
sealing four wells
317, are attached to each well containing element 348. That said, any suitable
arrangement or
number of bottom pieces may be used, e.g. all the wells in a single well
containing element
348 may be sealed with a single piece 334, each well 317 may be sealed with an
individual
bottom piece 334, or six bottom pieces 334 may be used to seal pairs of wells
317 in a single
well containing element 348. In some embodiments, frame 322 includes, adjacent
a bottom
portion thereof, partitions 350 which divide the frame into sections 352, each
section 352
being suitably sized to fit one of bottom pieces 334, and the wells 317
attached thereto.
It will be appreciated that when assembled together, the circumference of the
uppermost portion of the collection of well-supporting elements 330, including
the flanges
332, is slightly less than the inner circumference of frame 322, so that when
the flanges 332
of well-supporting elements 330 rest on blocks 328, there is a small gap 338
between the
well-supporting elements 330 and the frame 322 (see Fig. 3E), as well as
between elements
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330 themselves (see Fig. 3E). The presence of gaps 338 allows for movement of
the well-
supporting elements 330 along the vertical axis and for concomitant
deformation of arms 326,
as will be explained below. In order to seal the gaps 338 without inhibiting
such movement, a
very thin (for example, approximately 7 micron) film 340 of a flexible
material, such as a
suitable plastic, is attached to an upper edge 342 of the frame 322 and to at
least a portion of
the upper surface of well-supporting elements 330. Film 340 includes eight
rectangular
openings 343 which are aligned with the apertures 333 in the assembled well-
supporting
elements 330.
Film 340, well supporting elements 330, bottom pieces 334, and frame 322, are
attached to each other using any suitable means, such as soldering, adhering,
melting,
bonding, or any other suitable attachment mechanism. It will be appreciated
that in the
embodiment shown, well-containing elements 348 may be easily removed from well
supporting elements 330, without damaging the sensitive structure and
functionality of arms
326, which is described in further detail hereinbelow.
As noted, when plate 300 is assembled, well-supporting elements 330 rest on,
and are
attached at each of their longitudinal ends to, a pair of blocks 328. This
restricts the motion of
elements 330 in the lateral and longitudinal (x- and y-) directions, but
allows for motion in
the vertical (z-) direction. Thus, when fluid is placed into (or removed from)
a well in a well
containing element 348 located in any of well supporting elements 330, the
increase (or
decrease) in the weight of the fluid will result in the deformation of arms
326 attached to that
well supporting element 330. It will be appreciated that typically not more
than several
hundred microliters, and in some applications only a few microliters or less,
are added to a
given well 317. Consequently, the flexible arms 326 should be of suitable
flexibility to
deflect in response to the addition (or removal) of such quantities of fluid
(or the equivalent
masses) to (or from) the wells. For example, less than 300 microliters ( 1),
less than 250 1,
less than 200 1, less than 15 I, less than 100 1, less than 75 I, less
than 50 1, less than 45
1, less than 40 1, less than 35 1, less than 30 1, less than 25 pi, less
than 20 I, less than
15 1, less than 10 1, less than 5 1, less than 4 1, less than 3 1, less
than 2 I, or less than 1
1 of fluid may be added to a well 317 at any given time.
Attached to the upper and lower face of each arm 326 near the point at which
the arm
is attached to one of -stairs" 325 (which will themselves be described in more
detail below) is
a thin, flat strain gauge 344. It will be appreciated that alternatively,
rather than place a strain
gauge on the upper face and one on the lower face an arm, two strain gauges
may be placed
on the upper face of an arm, one near where the arm is attached to frame 322
and the other
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near where the arm is attached to block 328, or two strain gauges may be
placed on the lower
face of an arm only, one near where the arm is attached to frame 322 and the
other near
where the arm is attached to block 328.
Each strain gauge 344 is electrically coupled (e.g. by thin wires, not shown)
to a thin,
flat electronic card 346 located below lower arms 326. Preferably, electronic
cards 346 are
positioned so as to minimize the length of the connections between the strain
gauges 344 and
the cards. Cards 346 are electrically coupled, via a plurality of wires 356,
to a port 358
located on frame 322, which port is configured for connection to an electrical
port of a
suitably equipped plate base and data reader, as described hereinbelow with
reference to Figs.
8 to 9B. In some embodiments, the data reader may run a graphical user
interface as
described hereinbelow with reference to Figures 5A to 7. Preferably, port 358
does not extend
beyond, and preferably is flush with, the outer surface of frame 322, and does
not affect the
overall dimensions of plate 300, thus allowing use of standard equipment. In
some
embodiments, plate 300 further includes a power supply (not shown) such as a
rechargeable
battery, for example connected to electronic card 346. which power supply
provides power to
electronic components of plate 300 and may be recharged when plate 300 is
connected to a
power source or computation device via port 358 or a USB port.
Electronic cards 346 may have located thereon an element for measuring
electrical
resistance in a circuit, such as a Wheatstone Bridge. The deflection of the
arms 326 caused by
a change of the volume, or weight, contained in one or more of wells 317,
leads to a change
in the length of the resistors in corresponding strain gauges 344. This change
in strain gauges
344 is correlated to the change in electrical resistance in a circuit, which
change is measured
by the elements on electronic cards 346, using e.g. a Wheatstone Bridge. The
change in the
electrical resistance in the circuit allows for calculation of the mass of the
fluid added to (or
removed from) the wells 317. Specifically, a greater mass of fluid added to
(or removed
from) the wells, results in a greater change in the deflection of arms 326,
which in turn leads
to a greater change in the electrical resistance in the circuit. Thus,
measurement of changes in
the electrical resistance in the circuit, is indicative of, and allows for
calculation of the change
in the mass of fluid in the wells. If the density of the fluid is known, this
facilitates
computation of the volume of fluid added (or removed). As such, arms 326
together with
strain gauges 344 form signal providers, for providing signals indicative of a
change in the
amount of fluid in one or more of the wells.
For example, if columns A through L of plate 300 are filled sequentially using
an 8-
tip dispenser, it is possible to iteratively calculate the amount of fluid
added to each well in
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each column, and thereby to identify in real time, on a well-by-well basis,
when an incorrect
amount of fluid, either too much or too little, has been added to a particular
well. Preferably,
the apparatus used to add fluid to the wells will be equipped with control
software that will
allow the apparatus used to correct for the error. In the case in which too
little fluid has been
added, additional fluid may be dispensed to the affected well so as to reach
the correct
amount of fluid in the well, and/or the software may be able to adjust for the
error by adding
proportionately less reagent or reactant liquid to the affected well in later
manipulations.
Similarly, if too much fluid has been added to a particular well, the addition
of reagents or
reactants in further manipulations may be scaled up appropriately.
Alternately, the affected well, or well-containing element, may be included in
further
manipulations during the remainder of the experiment, and the results of the
particular well
may be used in the calculations at the end of the experiment by adjusting the
calculations to
account for the incorrect volume used while conducting the experiment.
As a further alternative, identification of a particular well as having had an
incorrect
amount of fluid added thereto allows that particular well to be discarded from
the calculations
at the end of the experiment, rather than discarding the results for the
entire row or column in
which the well is located, or for the entire plate.
It will also be appreciated that even if all the wells are filled
simultaneously, the use
of plate 300 in conjunction with appropriate software to identify in real time
the addition of
an incorrect amount of fluid to a specific well supporting element 330,
enables the user to
stop running experiments using wells in that particular element 330 or in the
plate 300, thus
avoiding waste of reagents, reactants and the like in downstream experiments.
Additionally, electronics cards 346 may have electrically coupled thereto
components
for manipulating data collected by the various sensor elements coupled to the
cards, such as
an analog-to-digital converting component for converting the analog signals of
the
Wheatstone Bridge to digital signals, and normalizing components for
normalizing the
collected signals.
In some embodiments, plate 300 also includes one or more temperature sensors
(not
shown), electrically coupled to electronics cards 346, and configured to
provide an indication
of the temperature, or of a temperature change, in the vicinity of one or more
of wells 317. It
is appreciated that a temperature change in the system may affect the strain
gauges 344, and
therefore knowledge of and computational consideration of changes to the
temperature can
allow for more accurate identification of the weight in a well and for
ensuring a stable
temperature of the sample in the well, which may be sensitive to temperature
changes.
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In accordance with some embodiments of the invention, plate 300 includes means
for
heating and, optionally, cooling individual wells. Such heating means may take
the form of,
for example. (a) a heating coil disposed around at least a portion of the
well. or (b) a Peltier
device, sometimes called a Peltier heat pump or thermoelectric cooler. As will
be appreciated,
a Peltier device can be used to cool as well as to heat an individual well, as
well as to sense or
monitor the temperature in the well or in the vicinity thereof. In this way,
the temperature in
individual wells may be controlled, for example the temperature in each well
may be
maintained at 37 C 0.5 C.
In some embodiments, a heating coil or Peltier device may be disposed on some
or all
of the well-supporting elements 330, for collectively heating two or more the
wells supported
by that well-supporting element. In other embodiments, heating coils or
Peltier devices may
be disposed on some or all of the individual wells 317, for example on
cylinders 320 thereof,
such that each heating coil or Peltier device heats a specific well 317 on or
in which it is
disposed.
It will also be appreciated that if the temperature of the wells is
periodically
measured, and if the device used to measure the temperature is coupled to a
controller that
controls the heating means for each individual well, then the individual well
heating means,
used in conjunction with the periodic measuring of temperature in individual
wells, can
provide a way to improve control over the conditions in a given well. Thus,
for example,
plates 300 having such heating means may be stored in an incubator and the
temperature of
the wells monitored periodically and the temperature of individual wells
adjusted, if
necessary, by heating (or cooling) individual wells. Alternatively, the
heating means may
themselves be used to effect incubation, for example temperature monitoring
and adjustment
may be effected frequently, say for example every 15, 10 or 5 minutes, to
maintain the
temperature in particular wells at e.g. 37 C 0.5 C so as to effect
incubation. Thus, by
equipping the plate 300, the well-supporting elements 330, or the wells 317
with individual
heating elements for each well, in cases in which it is found that the
temperature is incorrect,
the temperature may be adjusted and controlled.
Reference is now made to Fig. 3D, which is an enlarged perspective view of the
supports 324a and 324b, including arms 326 and blocks 328, and to Figures 3F
and 3G,
which are sectional illustrations taken along section lines IIIF-IIIF and IIIG-
IIIG respectively
in Fig. 3A. Figs. 3D, 3F, and 3G show in greater detail the supports 324a and
324b, the arms
326, the blocks 328, and the attachment of the arms 326 to the supports.
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As seen, eight blocks 328 are located near each longitudinal end of frame 322,
so that
each well supporting element 330 rests on and is attached to two blocks 328,
one at each
longitudinal end of element 330. In order to accommodate eight blocks at each
end of the
frame 322, the supports 324a and 324b and the blocks 328 are designed in a
step-stair
fashion, as described hereinbelow. A first support 324a protrudes from the
inner wall 322d of
one longitudinal end of frame 322 as well as from the longitudinal wall 322b
adjacent
thereto. At the other longitudinal end of frame 322, a second support 324a
protrudes from
longitudinal wall 322b but is spaced slightly from the inner wall 322a. In
both cases, in order
to accommodate four pairs of arms 326 and to facilitate appropriate
positioning of the blocks
328, support 324a is constructed in stair-step fashion, having two sets of
"stairs", an upper set
360a and a lower set 360b, formed in parallel. All of the "stairs" of support
324a protrude
from inner wall 322b of frame 322, but whereas all of the "stairs" have the
same width along
the longitudinal axis of frame 322, the uppermost "stairs" of each set, 325a
and 325a',
protrude the least from inner wall 322b, "stairs" 325b and 325b' protrude
slightly more,
"stairs" 325c and 325c' protrude more, and "stairs" 325d and 325d' protrude to
about
halfway between wall 322b and wall 322c.
The distance between the upper surfaces of each pair of corresponding -stairs"
(i.e.
between 325a and 325a', between 325b and 325b', between 325c and 325c', and
between
325d and 325d') is the same as the distance between the lower surfaces of each
pair of arms.
A first pair of arms 326a and 326a' is thus attached at the proximal ends
thereof to "stairs"
325a and 325a', a second pair of arms 326b and 326h' is attached at the
proximal ends
thereof to "stairs" 325b and 325b', a third pair of arms 326c and 326c' is
attached at the
proximal ends thereof to "stairs" 325c and 325c' and a fourth pair of arms
326d and 326d' is
attached at the proximal ends thereof to "stairs" 325d and 325d'. The "stairs"
are spaced
from each other such that the arms in each pair of arms are parallel to one
another, and so that
in the course of normal use, each pair of arms may move without contacting
another pair of
arms.
As seen in Figs. 3D and 3F, at their distal ends, the arms 326 are attached to
blocks
328, such that each pair of arms is attached in parallel to a single block
328. In order to
facilitate a compact arrangement of the arms, each block 328 comprises an
upper inner
surface for attachment of a first arm thereto, a lower inner surface for
attachment of a second
arm thereto, and a recess allowing the additional arms to pass therethrough.
Thus, arm 326a
attached to stair 325a is attached to an upper inner face 328a' of block 328a
which is closest
to the center of the frame, and block 328a is constructed with a recess 328a"
in order to allow
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arms 326b, 326c and 326d to pass therethrough without touching each other and
without
touching block 328a. Similarly, arm 326a' attached to stair 325a' is attached
at its distal end
to the bottommost surface 328a" of block 328a, with arms 326b', 326c' and
326d' passing
thereunder in parallel to one another. Block 328b is similarly constructed, to
allow for
attachment of arms 326b and 326b' at surfaces 328b' and 328b" respectively and
to allow
passage therethrough of arms 326c and 326d and passage of arms 326c' and 326d'
thereunder. Block 328c is constructed to allow for attachment of arms 326c and
326c' at
surfaces 328c' and 328c" and to allow passage therethrough of arm 326d and
passage of
arm 326d' thereunder. Block 328d, which of the four blocks 328a, 328b, 328c
and 328d is
closest to wall 322c, is constructed to allow attachment of arms 326d and
326d' at surfaces
328d' and 328d" respectively.
It will be appreciated that as a result of this construction, the sizes of the
upper portion
of blocks 328a, 328b. 328c and 328d differ from one another. It will also be
appreciated that
each of blocks 328a, 328b, 328c and 328d are of different overall height, and
while it is
preferable that the location of the uppermost surface of each block, relative
to the top of the
frame 322, be the same, there can be slight differences in the location of the
uppermost
surface of each block, as the software used to calculate the displacement and
thus the mass of
fluid added to the wells can be programmed to account for such differences.
For the same
reason, it is not necessary that all the blocks 328 have the same mass.
As seen clearly in Figure 3G, support 324b is arranged in an analogous manner
to that
of support 324a, in the opposite direction. At one longitudinal end of the
frame 322, a first
support 324b protrudes from the inner wall 322a of one longitudinal end of
frame 322 as well
as from the longitudinal wall 322c adjacent thereto. Al the other longitudinal
end of frame
322, a second support 324b protrudes from longitudinal wall 322c but is spaced
slightly from
inner wall 322d. Near wall 322d, support 324b is spaced therefrom so as to
accommodate
arms 326a, 326a', 326b, 326b', 326c, 326c', 326d and 326d'. In order to
accommodate four
pairs of arms 326 and to facilitate appropriate positioning of the blocks 328,
support 324b is
constructed in stair-step fashion, having two sets of "stairs", an upper set
362a and a lower
set 362b, formed in parallel. All of the -stairs" of support 324b protrude
from inner walls
322a (or 322d) and 322c of frame 322, but whereas all of the "stairs" have the
same width
and thus project the same distance from wall 322a (or 322d), the uppermost -
stairs" of each
set, 325e and 325e', protrude the least from inner wall 322c, "stairs" 325f
and 325f' protrude
slightly more, "stairs" 325g and 325g' protrude more, and "stairs" 325h and
325h' protrude
to about halfway between wall 322c and wall 322b.
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The distance between the upper surfaces of each pair of corresponding "stairs"
(i.e.
between 325e and 325e', between 325f and 325f'. between 325g and 325g', and
between
325h and 325h') is the same as the distance between the lower surfaces of each
pair of arms.
A first pair of arms 326e and 326e' is thus attached at the proximal ends
thereof to "stairs"
325e and 325e', a second pair of arms 326f and 326f' is attached at the
proximal ends thereof
to "stairs" 325f and 325f', a third pair of arms 326g and 326g' is attached at
the proximal
ends thereof to "stairs" 325g and 325g' and a fourth pair of arms 326h and
326h' is attached
at the proximal ends thereof to "stairs" 325h and 325h'. The "stairs" are
spaced from each
other such that the arms in each pair of arms are parallel to one another, and
so that in the
course of normal use, each pair of arms may move without contacting another
pair of arms.
As seen in Figures 3D and 3G, at their distal ends, the arms 326 are attached
to blocks
328, each pair of arms being attached in parallel to a single block 328. In
order to facilitate a
compact arrangement of the arms, each block 328 comprises an upper inner
surface for
attachment of a first arm thereto, a lower inner surface for attachment of a
second arm
thereto, and a recess allowing the additional arms to pass therethrough. Thus,
arm 326e
attached to stair 325e is attached to an upper inner face 328e' of block 328e
which is closest
to the center of the frame, and block 328e is constructed with a recess 328e"
in order to allow
arms 326f, 326g and 326h to pass therethrough without touching each other and
without
touching block 328e. Similarly, arm 326e' attached to stair 325e' is attached
at its distal end
to the bottommost surface 328e" of block 328e, with arms 326f', 326g' and
326h' passing
thereunder in parallel to one another. Block 328f is similarly constructed, to
allow for
attachment of arms 326f and 326f' at surfaces 3281" and 328f" respectively and
to allow
passage therethrough of arms 326g and 326h and passage of arms 326g' and 326h'
thereunder. Block 328g is constructed to allow for attachment of arms 326g and
326g' at
surfaces 328g' and 328g" and to allow passage therethrough of arm 326h and
passage of
arm 326h' thereunder. Block 328h, which of the four blocks 328e, 328f, 328g
and 328h is
closest to wall 322b, is constructed to allow attachment of arms 326h and
326h' at surfaces
328h' and 328h" respectively.
It will be appreciated that as a result of this construction, the sizes of the
upper portion
of blocks 328e, 328f, 328g and 328h differ from one another. It will also be
appreciated that
each of blocks 328e, 328f, 328g and 328h arc of different overall height, and
while it is
preferable that the location of the uppermost surface of each block, relative
to the top of the
frame 322, be the same, there can be slight differences in the location of the
uppermost
surface of each block, as the software used to calculate the displacement and
thus the mass of
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fluid added to the wells can be programmed to account for such differences.
For the same
reason, it is not necessary that all the blocks 328 have the same mass.
It will be appreciated that since reagents and other fluids are added into or
removed
from wells 317 in well-containing elements 348, which are removable from plate
300, plate
300 may in principle be used multiple times and/or for multiple experiments,
provided that
well-containing elements 348 are replaced between each use of the plate 300.
Reference is now made to Figs. 4A ¨ 4C, which show a multi-well plate 400 and
components thereof, constructed and operative in accordance with embodiments
of the
teachings herein. Specifically, Fig. 4A is a perspective view of the multi-
well plate 400. Fig.
4B is an exploded view of the multi-well plate 400, and Fig. 4C is a sectional
view of the
multi-well plate 400, taken along section lines IVC-IVC in Fig. 4A.
As seen in Fig. 4A, when assembled, plate 400 looks generally similar to plate
300 of
Figs. 3A-3G. However, it will also be apparent from Fig. 4B and from the
description herein
that although the purposes and uses of plates 300 and 400 are similar, the
construction of
plate 400 is somewhat different from that of plate 300.
Plate 400 is designed for use with existing equipment and is therefore sized
in
accordance with standard plate sizes currently in use, with its wells
similarly spaced; thus
when assembled, plate 400 looks similar to a typical 96-well plate, with an
upper surface 412,
a lower surface 414, a plurality of sides 416 between surfaces 412 and 414,
and having a
plurality of wells 417 formed therein.
As shown in Fig. 4B, which is an exploded view of plate 400, plate 400 is
actually
formed of several parts. Sides 416 are part of a frame 422, which also
includes a well-
defining skeleton 450, which defines 96 bores 452 in frame 422, each bore
having a generally
circular cross section along the horizontal plane, and a generally rectangular
cross section
along the vertical plane.
Plate 400 further includes 96 individual identical cylindrical well supporting
elements
430, each including an aperture 433, each sized to fit in one of bores 452 and
suited to receive
a single well-defining element 419, which may be easily removed from a well
supporting
element 430 in which it is accommodated. Each individual well 417 is defined
in by well-
defining element 419 which is formed of a cylindrical portion 466 and is
sealed at its bottom
with a generally circular bottom piece 434.
Each well-supporting element 430 is adhered to, or otherwise attached to, two
arms
426 and 426' located in plates 474 and 476, respectively. Arms 426 and 426'
may be, but are
not necessarily, formed integrally in plates 474 and 476. For ease of
reference, henceforth
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arms 426 and 426' will be referred to as 426, unless specifically noted
otherwise. The flexible
arms 426 may be made of a suitable material, generally metal or plastic, which
is suitably
flexible as to be sensitive to the addition or removal of a small volume to or
from the wells,
as will be explained in more detail below.
Arm 426 is generally flat and, as shown in the inset in Fig. 4B, each arm 426
and 426'
has a generally rectangular portion 426a from which extend, at two adjacent
corners thereof,
protrusions, which form a partial annulus 426b that extends more than half-way
but not
completely around cylindrical well supporting element 430, and is sized to
have an inner
circumference slightly larger than the outer circumference of cylindrical well
supporting
element 430. Within partial annulus 426b is formed a ring 426c, which is sized
to have the
same inner circumference as cylindrical well supporting element 430.
Cylindrical well
supporting element 430 is thus affixed to ring 426c (the upper surface of
cylindrical well
supporting element 430 being attached to the lower surface of the ring 426c of
arm 426 and
the lower surface of cylindrical well supporting element 430 being attached to
the upper
surface of the ring 426c of arm 426'). for example by an adhesive. This
construction enables
a pair of arms 426 and 426' to hold well supporting element 430 in place in
the x- and y-axes
but allows movement of the element and the well contained therein along the z-
axis, with
concomitant bending of arms 426.
In order to maximize and localize the strain felt by the arms at rectangular
portions
426a and partial annuluses 426b, the partial annuluses 426b and rectangular
portions 426a
are formed of a suitably thin and flexible material, such as metal or plastic.
In some
embodiments, to facilitate bending, arms 426 and 426' are made thinner than
the rest of
plates 474 and 476, respectively. In some embodiments, portions of plates 474
and 476
exterior to arms 426 and 426' are adhered, or otherwise attached, to frame 422
and/or to an
electronic card 446 (described in more detail hereinbelow), so as to
strengthen portions of the
plates 474 and 476 which need not bend, thereby localizing the strain felt by
the arms 426.
It will be appreciated that typically not more than several hundred
microliters, and in
some applications only a few microliters or less of fluid are added to a given
well. For
example, less than 300 microliters (pi), less than 250 1, less than 200 1,
less than 150 1,
less than 100 1, less than 75 1, less than 50 1, less than 45 1, less than
40 1, less than 35
I, less than 30 I, less than 25 I, less than 20 I, less than 15 I, less
than 10 I, less than 5
1, less than 4 I, less than 3 I, less than 2 1, or less than 1 I may be
added to a given well.
Consequently, the flexible arms 426 should be of suitable flexibility to
deflect in response to
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the addition (or removal) of such quantities of fluid (or the equivalent
masses) to (or from)
the wells.
When assembled together, the outer circumference of cylindrical well
supporting
elements 430 is slightly smaller than the inner circumference of bores 452,
thereby forming
small gaps 438 between the well-defining skeleton 450 and the cylindrical well
supporting
elements 430 (see Fig. 4C). The presence of these gaps allows for independent
movement of
each well-supporting element 430 along the vertical (z-) axis and concomitant
deformation of
arms 426, as will be explained below.
In order to seal the gaps 438 without inhibiting such vertical movement, a
very thin
(e.g. approximately 7 micron) film 440 of a flexible material, such as a
suitable plastic, is
attached to the upper edge 442 of the frame 422, and in some embodiments also
to an upper
card 446 (described in more detail hereinbelow). Film 440 includes 96 circular
openings 443
which are aligned with the apertures 447 in cards 446 (described hereinbelow),
with rings
426d discussed in more detail below, and with apertures 433 in the assembled
well-
supporting elements 430.
Film 440 is attached to frame 422 using any suitable means, such as soldering,
adhering, melting, bonding, or any other suitable attachment mechanism. It
will be
appreciated that in the embodiment shown, each well-defining element 419 may
be easily
removed from the well-supporting element 430 in which it is positioned,
without damaging
the sensitive structure and functionality of arms 426.
Each arm 426 has a pair of flat strain gauges 444 attached alongside one
another on
the upper face thereof at the rectangular portion 426a. Alternately, the
strain gauges may be
attached to upper and lower faces of rectangular portion 426a. Each strain
gauge is
electrically coupled, for example via wires (not shown), to an electronic card
446. In some
embodiments, arms 426 may be formed with holes 445 to facilitate the passage
of such wires
therethrough. Electronic cards 446 are positioned so as to minimize the length
of the
connections between the strain gauges 444 and the cards.
In some embodiments, such as that shown in an enlarged portion of Figure 4B,
which
in order to shown certain details is rotated 90 degrees about a longitudinal
axis of the well-
supporting element relative to the rest of the Figure 4B, well-supporting
elements 430 may
include protecting protrusions 431a at top and bottom rims thereof, which
protecting
protrusions are designed to protect strain gauges 444. Additionally, in some
embodiments,
well-supporting elements 431 may include top and bottom protrusions 431b,
rotationally
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offset from protrusions 431a by 180 degrees, which protrusions 431b engage
plates 474 and
476 and limit the range of deflection of arms 426.
In some embodiments, cards 446 (each of which may actually be a plurality of
cards)
are electrically coupled, via a plurality of wires (not shown) to a port 458
located on frame
422, which port is configured for connection to an electrical port of a
suitably equipped plate
base and data reader, as described hereinbelow with reference to Figures 8 to
9A. In some
embodiments, cards 446 (each of which may actually be a plurality of cards)
are electrically
coupled, via a plurality of wires (not shown) to a USB port (not shown), or to
a similar
input/output port such as is presently known or as may be developed in the
future, located on
frame 422, which port is configured for connection of plate 400 to a power
source and/or to a
computation device. The data reader and/or the computation device to which
plate 400 may
be connected may run a Graphical User Interface as described hereinbelow with
reference to
Figures 5A to 7. Preferably, port 458 and/or the USB port (not shown) does not
extend
beyond, and preferably is flush with, the outer surface of frame 422 and does
not affect the
overall dimensions of plate 400, thus allowing use of standard equipment.
In some embodiments, plate 400 further includes a power supply (not shown)
such as
a rechargeable battery, for example connected to electronic card 446, which
power supply
provides power to electronic components of plate 400 and may be recharged when
plate 400
is connected to a power source or computation device via port 458 or a USB
port.
Each electronic card 446 includes 96 apertures 447 having a circular cross-
section,
such that when plate 400 is assembled, apertures 447 are aligned with rings
426d, with
sections 452. and with well supporting elements 430. Arranged near apertures
447 are an
additional ninety-six smaller apertures 448. Apertures 448 allow for the
passage of wires (not
shown) connecting the strain gauges 444 to cards 446.
Electronic cards 446 may have located thereon an element for measuring
electrical
resistance in a circuit, such as a Wheatstone Bridge. The deflection of the
arms 426 leads to a
change in the length of the resistors in corresponding strain gauges 444,
which is correlated to
the change in electrical resistance in a circuit, which change is measured by
the elements on
electronic cards 446, using e.g. a Wheatstone Bridge. The change in the
electrical resistance
in the circuit allows for calculation of the mass of the fluid added to (or
removed from) each
well. Specifically, a greater mass of fluid added to (or removed from) the
well, results in a
greater change in the deflection of arms 426, which in turn leads to a greater
change in the
electrical resistance in the circuit. Thus, measurement of changes in the
electrical resistance
in the circuit is indicative of a change in the mass of fluid in the well, and
allows for
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calculation of this change. If the density of the fluid is known, this
facilitates computation of
the volume of fluid added (or removed). As such, arms 426 together with strain
gauges 444
form a signal provider, for providing a signal indicative of a change in the
amount of fluid in
a well associated therewith. In some embodiments, the electronic cards 446
include a storage
component, for storing signals generated by the signal provider, for example
when the plate
400 is not connected to a data reader such as the data reader of Figure 8.
Such stored signals
may then be retrieved from the cards 446 by a data reader when the plate 400
is connected
thereto.
For example, if rows A through L of plate 400 are filled sequentially using an
8-tip
dispenser, or if individual wells are filled sequentially, or even if all the
wells are filled
simultaneously, it is possible to calculate the amount of fluid added to each
well, and thereby
to identify in real time, on a well-by-well basis, when an incorrect amount of
fluid, either too
much or too little, has been added to a particular well.
Preferably, the apparatus used to add fluid to the wells will be equipped with
control
software that will allow the apparatus used to correct for the error. In the
case in which too
little fluid has been added, additional fluid may be dispensed to the affected
well so as to
reach the correct amount of fluid in the well, and/or the software may be able
to adjust for the
error by adding proportionately less reagent or reactant liquid to the
affected well in later
manipulations. Similarly, if too much fluid has been added to a particular
well, the addition of
reagents or reactants in further manipulations may be scaled up appropriately.
Alternately, the affected well, or well containing element, may be included in
further
manipulations during the remainder of the experiment, and the results of the
particular well
may be used in the calculations at the end of the experiment by adjusting the
calculations to
account for the incorrect volume used while conducting the experiment. As a
further
alternative, identification of a particular well as having had an incorrect
amount of fluid
added thereto allows that particular well to be discarded from the
calculations at the end of
the experiment, rather than discarding the results for the entire row or
column in which the
well is located, or for the entire plate.
It will also be appreciated that even if all the wells are filled
simultaneously, the use
of plate 400 in conjunction with appropriate software to identify in real time
the addition of
an incorrect amount of fluid to a specific well supporting element 430,
enables the user to
stop running experiments using the well in that particular element or in the
plate 400, thus
avoiding waste of reagents, reactants and the like in downstream experiments.
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It will further be appreciated that the plate 400 may also operate without
being
continuously monitored. In such cases, a baseline measurement of a well or of
the plate is
obtained from the signal provider. Subsequently, the plate may be disconnected
from the
power source and/or the data reader or processor to which the signals are
provided, and fluid
added to or removed from the plate. The plate may then be reconnected to the
power source
and/or the data reader or processor, and a second signal obtained from the
signal provider.
Comparison of the initial and second signals enables identification of
specific wells where an
incorrect amount of fluid is present, allowing for those wells to be discarded
from further
experiments and computations.
In some embodiments, plate 400 also includes one or more temperature sensors
(not
shown), electrically coupled to electronics cards 446, and configured to
provide an indication
of the temperature, or of a temperature change, in the vicinity of one or more
of wells 417. It
is appreciated that a temperature change in the system may affect the strain
gauges 444, and
therefore knowledge of, and computational consideration of changes to the
temperature can
allow for more accurate identification of the weight in a well and for
ensuring a stable
temperature of the sample in the well, which may be sensitive to temperature
changes.
Additionally, electronics cards 446 may have electrically coupled thereto
components
for manipulating data collected by the various sensors elements coupled to the
cards, such as
an analog-to-digital converting component for converting the analog signals of
the
Wheatstone Bridge to digital signals, and normalizing components for
normalizing the
collected signals.
Because the deflection of each arm can be calculated, and thus the amount of
material
or volume of liquid added to each individual well can be calculated in real
time, the use of
plate 400 facilitates the correcting of the amount of material to be added to
each well, or the
ignoring of an individual well, rather than a row of wells or the whole plate,
in further
experimental manipulations. Also, with this configuration it is possible to
observe loss of
material from a given well over time, as will be described hereinbelow with
reference to
Figures 5A-7.
In accordance with some embodiments of the invention, plate 400 includes means
for
heating and, optionally, cooling individual wells. Such heating means may take
the form of,
for example. (a) a heating coil disposed around at least a portion of the
well. or (b) a Peltier
device, sometimes called a Peltier heat pump or thermoelectric cooler. As will
be appreciated,
a Peltier device can be used to cool as well as to heat an individual well, as
well as to sense or
monitor the temperature in the well or in the vicinity thereof. In this way,
the temperature in
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individual wells may be controlled, for example the temperature in each well
may be
maintained at 37 C 0.5 C.
In one embodiment, a Peltier device for each well may be built into one or
both
electronics cards 446 shown in Fig. 4B, for example adjacent each aperture 447
for heating of
a specific well-supporting element 430 or well-defining element 419 disposed
in the aperture.
Alternatively, a heating coil or Peltier device may be disposed on some of or
on each of the
well-supporting elements 430 or the well-defining elements 419, for heating
the well
associated therewith or the interior thereof. As a further alternative, a
heating coil or a Peltier
device may be disposed adjacent a group of well-supporting elements 430 or
well-defining
elements 419, for example on electronic card 446, for heating the wells in the
group or the
interiors thereof. Although reference is made to Fig. 4B, it will be
appreciated that the
provision of such well heating means is not limited to plates in which the
wells are
displaceable along the z-axis, and that such heating means may be provided in
plates in which
the wells are not displaceable.
It will also be appreciated that if the temperature of the wells is
periodically
measured, and if the device used to measure the temperature is coupled to a
controller that
controls the heating means for each individual well, then the individual well
heating means,
used in conjunction with the periodic measuring of temperature in individual
wells, can
provide a way to improve control over the conditions in a given well. Thus,
for example,
plates 400 having such heating means may be stored in an incubator and the
temperature of
the wells monitored periodically and the temperature of individual wells
adjusted, if
necessary, by heating (or cooling) individual wells. Alternatively, the
heating means may
themselves be used to effect incubation, for example temperature monitoring
and adjustment
may be effected frequently, say for example every 15, 10 or 5 minutes, to
maintain the
temperature in particular wells at e.g. 37 C 0.5 C so as to effect
incubation. Thus, by
equipping the plate 400, the well-supporting elements 430, or the well-
defining elements 419
with individual heating elements for each well, in cases in which it is found
that the
temperature is incorrect, the temperature may be adjusted and controlled.
It will be appreciated that since reagents and other fluids may be added into
or
removed from well-defining elements 419, which may be removed from plate 400
and
optionally disposed of, plate 400 may be used multiple times and/or for
multiple experiments,
provided that well-defining elements 419 are replaced between each use of the
plate 400.
32
Reference is now made to Figures 5A to 5D, which are screen shots illustrating
a graphical user interface for on-line (real-time) monitoring of addition of
fluid to a
multi-well plate in accordance with embodiments of the teachings herein.
As described hereinabove, multi-well plates in accordance with embodiments
of the present invention, such as multi-well plate 300 of Figures 3A to 3G and
multi-
well plate 400 of Figures 4A to 4C, may be electrically coupled to a
processor, for
example via a suitably equipped plate data reader, as described hereinbelow
with
reference to Figures 8 to 9B, or via a USB or other cable connected to a
computation
device such as a computer. The graphical user interface of Figures 5A to 5D
runs on
such a processor coupled to the plate, using data provided to the processor
from the
electronics cards of the plates (such as electronics cards 346 and 446). The
processor
may be configured to provide only graphical user information, or it may also
be
configured to control the amount fluid dispensed into the wells.
It will be appreciated that the data for providing online monitoring depends
on
measurement of a baseline electrical resistance measured by the strain gauges
on the
plate (such as strain gauges 344 and 444), which baseline measurement when
monitoring the filling of a well generally corresponds to an empty well. Once
fluid is
dispensed into the well, the electronics cards provide to the processor an
indication of
the volume of fluid added into each well, thereby facilitating the function of
the
graphical user interface as described hereinbelow. In some embodiments, the
analog
data representing the baseline electrical resistance is normalized and
converted to
digital data by suitable elements located on the electronics cards of the
plate, such that
the processor receives data suitable for use in the graphical user interface.
As seen, a graphical user interface 500 is associated with experiment planning
software (not shown), such that the specific details of the experiment being
conducted,
such as the experiment name, the material used, the volume(s) of liquid(s) to
be
dispensed, the volume upper and lower limits, the required accuracy (i.e. the
experimental sensitivity), and any other suitable experimental parameters are
displayed
in an information box 502 of the graphical user interface. The details
included in
information box 502 provide an indication to the user of the criteria that
will be used to
alert the user of incorrect addition of material to any one or more wells, as
described
hereinbelow. It will be appreciated that in some embodiments, the software
running the
apparatus dispensing the fluid is able to calibrate quantities to be dispensed
and the
degree of sensitivity to be used based on these experimental parameters.
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Graphical user interface 500 further includes a graphic representation 504 of
a multi-
well plate in which the experiment is currently being conducted, based on
information
provided from the electronics card of the plate. Graphic representation 504 of
the plate
includes a plurality of circles 506, each corresponding to a well in the plate
(here shown as a
96 well plate), as well as indications of the rows and columns of the plate,
indicated by
reference numerals 508 and 510, respectively.
The purpose of online monitoring of addition of fluid to the plate is to
facilitate real
time control of the volume of fluid added to the plate. The graphic user
interface 500 allows a
user to monitor the situation in real time and, in cases in which control of
the process is not
completely automated after the initial inputting of parameters, to instruct
the system as
necessary to correct the fluid volume or take other steps to compensate for an
incorrect
volume in a given well. Typically, the graphic user interface additionally
provides a graphic
indication whether the target volume of fluid has been reached, or whether
additional fluid
should be added to the plate. In some embodiments, such as that shown in
Figures 5A to 5D,
the graphic indication comprises a fill pattern or color indication, such that
a first fill pattern
or color represents an empty well, a second fill pattern or color represents a
well in which the
volume of fluid is less than the target volume of fluid, and a third fill
pattern or color
represents a well in which the volume of fluid is correct and is equal to the
target volume of
fluid, within the specified tolerance. In some embodiments, a fourth fill
pattern or color is
used to represent a well in which the target volume of fluid has been
exceeded.
Figure 5A illustrates the graphical user interface 500 prior to the beginning
of the
experiment. As such, all the circles 506 have no fill pattern (or are in a
first color), which is
indicative of an empty well.
Figure 5B illustrates the graphical user interface 500 when dispensation of
fluid into
column 1 of the plate has begun. As seen, in graphic representation 504, the
circles 512
corresponding to the wells of column 1 (wells Al, Bl, Cl, D1, El, Fl, Gl, and
Hp have a
second fill pattern (or are in a second color), here shown as diagonal lines
sloping from right
to left, indicative of a volume of fluid, which is less than the target
volume, being in the
wells, while the remaining circles 506, corresponding to wells in columns 2-
12, remain in the
pattern or color indicating empty wells.
In some embodiments (not shown), graphic representation 504 indicates the
volume
of fluid that must be added to a well in order to reach the fluid target
volume in that well. For
example, this may be achieved by scrolling a pointer, such as is controlled by
a computer
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mouse, over one of circles 506 resulting in a pop-up box indicating the volume
that should be
added to the corresponding well.
In Figure 5C it is seen that as dispensation of fluid into the wells of column
1
continues, most of the circles 512, corresponding to wells in which the target
volume of fluid
has been reached, are represented in a third fill pattern (or are in a third
color), here shown as
dense diagonal lines sloping rom left to right, indicative of reaching the
target volume. The
graphic representation 504 additionally indicates that in well Cl the target
volume of fluid
has not yet been reached, by maintaining circle 514, corresponding to well Cl,
in the second
fill pattern of diagonal lines sloping from right to left (or in the same
color).
Alternately, in embodiments in which the amount of fluid in the wells is
determined
only after completion of dispensation of fluid into a well (as opposed to
making continuous
or multiple determinations as the well is filled, for example if liquid is
dispensed
continuously but slowly or is dispensed drop-wise), graphic representation 504
does not
provide information such as that shown in Figure 5B. Rather, once the fluid
has been
dispensed, graphic representation 504 indicates in which wells the volume of
fluid falls short
of (or exceeds) the required volume, in a manner similar to that shown in
Figure 5C.
Figure 5D is identical to Figure 5C, but illustrates the graphical user
interface
following the dispensation of additional volume fluid into well Cl so as to
correct the initial
shortfall, such that the volume of fluid in well Cl is equal to the target
volume for the
experiment. As all the wells of column I are now correctly filled with the
target volume of
fluid, the corresponding circles 512 are represented with the third fill
pattern of dense
diagonal lines sloping from left to right (or in the third color), indicating
that the target
volume has been reached.
As fluid is dispensed into wells in additional columns of the plate, the
graphic
representation 504 changes, such that the color of each circle 506 is
indicative of the volume
of fluid in the corresponding well, thereby providing a real-time indication
of the volume of
fluid in each well, and assisting in preventing errors in the volume of fluid
added to the wells.
In some embodiments, fluid is dispensed into all the wells 506 of the plate
simultaneously, whether incrementally, continuously slowly or continuously
quickly. In such
embodiments, graphic representation 504 provides indications, similar to those
shown in
Figures 5B, 5C, and 5D, for all the wells at once, rather than row by row as
described
hereinabove.
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Reference is now made to Figures 6A and 6B, which are screen shots
illustrating a
graphical user interface for off-line volume monitoring of fluid in a multi-
well plate in
accordance with embodiments of the teachings herein.
Graphical user interface 600 of Figures 6A and 6B is similar to graphical user
interface 500 of Figures 5A to 5D, in that it runs on the processor coupled to
the plate, using
data provided to the processor from electronics cards of the plates (such as
electronics cards
346 and 446) via a plate data reader or a USB or other connector as is
presently known or
may be developed in the future. Similarly, graphical user interface 600
includes a graphic
representation 604 of a multi-well plate in which the experiment is currently
being conducted,
based on information provided from the electronics card of the plate. Graphic
representation
604 of the plate includes a plurality of circles 606, each corresponding to a
well in the plate
(here shown as a 96 well plate), as well as indications of the rows and
columns of the plate,
indicated by reference numerals 608 and 610, respectively.
However, during off-line monitoring, the purpose is not to indicate to the
user
whether or not an appropriate volume of fluid has been dispensed into a well,
but rather to
provide an alarm if, for some reason, the volume of fluid in a well has
dropped below the
target volume, or below a predetermined threshold value. As such, graphical
user interface
600 is not associated with experiment planning software, but rather has
inputted thereto
values at which the user should receive an indication that the volume in a
well is
inappropriate, such as an alarm or an audible or text notification to take
corrective action. In
some embodiments, the values are predetermined absolute volume values, such
that when the
volume of fluid in a well drops below the predetermined volume, the graphical
user interface
600 provides an indication of a specific well in which the volume is low. In
some
embodiments, such as the illustrated embodiment, the values are volume change
values, such
that the graphical user interface 600 provides an indication when the volume
of fluid in a well
changes by more than a predetermined value. In a variation on this (not
shown), an indication
may be given when the volume falls by more than certain percentage below a
predetermined
baseline value. The values used for providing an indication to the user may be
default values,
or may be set by the user based on the sensitivity of the experiment, or based
on other
considerations as suitable.
As seen in Figures 6A and 6B, volume changes of different magnitudes are
indicated
by different fill patterns or colors, and a legend 612 is provided so that the
user can identify,
based on the fill pattern or color of a circle 606, how much fluid is missing
from the
corresponding well. In the illustrated embodiment a fill pattern of diagonals
indicates that the
36
volume of fluid in the corresponding well is unchanged, and is equal to the
initial volume, a dotted
fill pattern indicates a change of less than one microliter in the volume of
fluid in the corresponding
well, and a checkered fill pattern indicates a change of at least 1 microliter
but less than three
microliters in the volume of fluid in the corresponding well.
Graphical user interface 600 further includes one or more graphs 614, in which
the volume of
fluid in one or more specific wells may be plotted as a function of time. In
some embodiments, the
specific well for which information is displayed in graph 614 may be selected
by the user, for example
by pointing the cursor of the mouse to a specific well or by typing the well
identification in a suitable
text box (not shown). In some embodiments, information corresponding to each
of the wells may be
displayed sequentially and/or repeatedly in graph 614.
Typically, the data reader or USB or other connector associated with the
electronics cards of
the plate reports the measured volume in each well to the processor at a fixed
rate, for example once
every day, once every hour, once every minute, once every half a minute, or
even once every second.
The exact rate may be factory coded in the electronics card, or may be set by
the user in accordance
with the demands of the experiment being conducted.
Turning to Figure 6A, it is seen that at a first time point Ti, there is no
change in the volume
of fluid in any of the wells, and thus all of circles 606 are filled in the
fill pattern corresponding to the
nominal volume, diagonal lines. Since there is no change in the volume of
fluid in any of the wells,
no plot is presented on graph 614.
In Figure 6B, which illustrates the graphical user interface at a second time
point T2 later than
T1, it is seen that circles 616 corresponding to wells Cl, DI, and El are
filled in the fill pattern
indicating a change of less than 1 microliter in the volume of fluid in the
wells (dots), and circles 618
corresponding to wells A4, Bl, F1, and G1 are filled in the fill pattern
indicating a change of at least
1 microliter but less than 3 microliters in the volume of the fluid in the
wells (checkered). In the
illustrated example, graph 614 depicts a plot 620 of the change in volume in
well A4 as a function of
time, based on multiple readings of the volume of fluid in well A4.
Reference is now made to Figure 7, which is a screen shot illustrating a
graphical user
interface for off-line temperature monitoring of fluid in a multi-well plate
in accordance with
embodiments of the teachings herein.
Graphical user interface 700 of Figure 7 is analogous to graphical user
interface 600 of Figures
6A and 6B in that it includes a graphic representation 704 of a multi-well
plate in which the
experiment is currently being conducted, including a plurality of circles 706,
each corresponding to
a well in the plate (here shown as a 96 well plate), as well as indications of
the rows and columns of
the plate, indicated by reference numerals 708 and 710, respectively. However,
graphic user interface
37
CA 2931068 2017-09-21
700 differs from graphical user interface 600 in that the fill patterns (or
colors) of circles 706 represent
a temperature of a corresponding well in the plate, as measured by one or more
temperature sensors
forming part of the plate, and in that one or more graphs 714 may include a
plot of the temperature in
one or more specific wells as a function of time.
In some embodiments (such as shown in Fig. 7) there is a temperature sensor
associated with
each well, whereas in other embodiments there may be fewer temperature sensors
than wells but still
multiplicity of temperature sensors.
As seen in Figure 7, different temperatures are indicated by different fill
patterns (or colors),
and a legend 712 is provided so that the user can identify, based on the fill
pattern (or color) of a
circle 706, what the temperature is in, or in the vicinity of, the well.
In Figure 7 it is seen that at a time point TI, each well has a specific
temperature as indicated
by the fill patterns of the corresponding circle 706. For example, the fill
pattern of circle 706
corresponding to well El indicates that the temperature of well El is 39 C.
In the illustrated example graph 714 includes a plot 720 showing the change in
temperature
at well A5 as a function of time.
Typically, the data reader or USB or other connector associated with the
temperature sensor(s)
of the plate reports the temperature in each well or in the vicinity of each
sensor to the processor at a
fixed rate, for example once every day, once every hour, once every minute,
once every half a minute,
or even once every second. The exact rate may be factory coded in the
electronics card, or may be set
by the user in accordance with the demands of the experiment being conducted.
As such, the fill
patterns (or colors) of circles 706 in graphical user interface 700 change
when the data reader indicates
a change in the temperature of the corresponding wells, as measured by the
temperature sensor(s).
It will appreciated that since well-containing elements 348 and well-defining
elements 419
are removable from their respective plates 300 and 400, it is possible to
obtain measurements using
elements 348 or elements 419, as described above, and then to remove elements
348 or elements 419
and insert them into other, simpler plates (not shown) which lack the
detecting means detailed above,
for storage, for example for storage in a refrigerator or incubator. At a
later time, if another
measurement is desired, elements 348 or elements 419 may re-inserted into
plate 300 or 400,
respectively.
Reference is now made to Fig. 8, which is a perspective view of a plate base
and data reader
800 constructed and operative in accordance with an embodiment of the
teachings herein, for
receiving signals from a multi-well plate in accordance with embodiments of
the invention.
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As seen in Figure 8, the base plate and data reader 800 includes a base 802
having
formed thereon a frame 810, suitably shaped and sized for receipt therein of a
multiwell plate,
such as plate 200, 300, or 400 described hereinabove. In some embodiments, the
base plate
and data reader 800 may form part of an optical instrument or imaging device,
such as. for
example, the Hermes system (http://www.idea-bio.com/page-87- Hermes.aspx)
commercially available from Idea Bio-Medical Ltd. of Rehovot, Israel. In some
such
embodiments, the base 802 may be transparent to at least some wavelengths of
illumination,
so as to allow for imaging of samples in the multi-well plate by the optical
instrument or
imaging device while a plate is disposed in the data reader 800.
In some embodiments, frame 810 includes a retaining mechanism for retaining
the
plate stable and immobile within data reader 800. In some embodiments, the
retaining
mechanism comprises protrusions 812 which engage the frame of the plate, which
protrusions 812 may be retractable into frame 810, for example under the force
of a spring.
As such, when a user inserts the plate into data reader 800, the user pushes
the plate against
the protrusions 812, causing the protrusions 812 to retract into frame 810.
Once the user stops
pushing the plate, for example when the plate is in place, the springs push
protrusions 812
outward such that protrusions 812 engage the plate and retain it within data
reader 800. In
some embodiments, the retaining mechanism may comprise a mechanism for snap-
fitting the
plate into place on data reader 800, a rim on which the plate may rest, and
the like.
In some embodiments, frame 810 includes indentations 814 to assist the user in
gripping the plate disposed within frame 810 when the user wishes to remove
the plate. Other
mechanisms for assisting in removal of the plate from frame 810, such as an
eject button,
may also be used.
Frame 810 additionally includes an electrical port 820, positioned and
configured to
electrically engage a corresponding port on the plate disposed within the data
reader, for
example such as port 358 of Fig. 3B or port 458 of Figure 4B. Electrical port
820 is also
electrically connected to a processor (not shown) for provision of information
from the plate
to the processor, for example for use of dedicated software such as experiment
planning
software or Graphical User Interface software as described hereinabove with
reference to
Figs. 5A to 7.
Reference is now made to Figs. 9A and 9B, which are perspective views of a
device
for removing well-containing elements or well-defining elements from and/or
for emplacing
such elements in a multi-well plate, the device constructed and operative in
accordance with
an embodiment of the teachings herein.
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As seen in Figs. 9A and 9B, a device 900 for manipulation of well containing
elements such as elements 330 or of well defining elements such as elements
419 is
functionally associated with a plate-bearing base 902 arranged to have a multi-
well plate 904
disposed therein. The plate bearing-base 902 may be a data reader and base
such as data
reader and base 800 described hereinabove, or may be a simple base on which a
multiwell
plate rests, as illustrated in Figures 9A and 9B.
Arranged above plate-bearing base 902 is a well-engaging portion 906 which is
movably mounted onto a vertical displacement mechanism 908. Vertical
displacement
mechanism 908 is configured to enable vertical displacement of well-engaging
portion 906
toward and away from a plate 904 disposed on plate-bearing base 902. In some
embodiments,
vertical displacement mechanism 908 includes vertical mounts 910 and a
displaceable portion
912 vertically displaceable along mount 910, such that said well engaging
portion 906 is
mounted onto displaceable portion 912 and is displaceable therewith.
Disposed on a lower surface 914 of well engaging portion 906 are a plurality
of well-
engaging protrusions 916, each configured to fit in one of the wells of a well-
containing
element or a well-defining element to be placed in plate 904 or being removed
from plate 904
for attachment thereto. In some embodiments, well-engaging protrusions 916
engage the
corresponding wells by snap fit mechanism, though other methods of engagement,
such as by
vacuum, are also considered.
For placement of well-defining elements or well-containing elements in plate
904,
well-engaging protrusions 916 engage the well-defining elements or well-
containing
elements, and subsequently well engaging portion 906 is vertically displaced
toward plate
904 until the well defining elements or well-containing elements are fitted in
their suitable
locations within plate 904, such as within sections 352 of plate 300 or within
well-supporting
elements 430 of plate 400. The well engaging protrusions 916 then disengage
from the well-
defining elements or well-containing elements and well engaging portion 906 is
vertically
displaced away from plate 904, leaving the elements properly placed within
plate 904 and
accessible for insertion of reagents thereinto.
For removal of well-defining elements or well-containing elements from plate
904,
well-engaging portion 906 is vertically displaced toward plate 904 until the
well-defining
elements or well-containing elements located within plate 904 engage to well-
engaging
protrusions 916. Well-engaging portion 906 together with well-engaging
protrusions 916 and
the wells engaged therewith are vertically displaced away from plate 904,
resulting in
removal of the well-defining elements or well containing elements from their
locations within
plate 904. When the well-engaging portion is sufficiently displaced from plate
904, the well-
engaging protrusions 916 then disengage from the well-defining elements or
well-containing
elements.
It is appreciated that a similar device may be used for engaging pipette tips
or the like,
and for dispensing fluids such as reagents into the wells in plate 904.
Described hereinbelow are variations in construction that may employed with
multiwell
plates such as those herein described with reference to Figures 2A to 4C, and
in some cases
with other multiwell plates, as well as variations in the methods of using
those plates.
The plates described above utilize the physical displacement of wells along
the z-axis
to determine the volume of fluid dispensed into one or more wells. In the
embodiments
described above, such physical displacement is coupled to strain gauges, in
order to induce a
signal that is correlated to the amount of displacement and thus the volume of
fluid dispensed
into (or lost from) the well(s) under observation (given that a fluid of a
known density and mass
occupies a determinable volume). However, it will be appreciated that other
methods may be
employed instead of or in addition to the use of strain gauges to determine
volume.
Thus, for example, if a multiwell plate having wells which are displaceable in
the z-
axis is used in conjunction with a reading device that has an auto-focus
mechanism, this may
be used to determine the amount of displacement of a well. An example of such
an auto-focus
mechanism is described in US Patent No. 7,109,459, entitled "Auto-focusing
method and
device for use with optical microscopy".
To illustrate, a multiwell plate having displaceable wells may be introduced
into a
reading device having an auto-focus mechanism, such as the WiscanTM scanner
available from
Idea Bio-Medical Ltd., Rehovot, Israel. By combining the auto-focus mechanism
with
appropriate feedback controls, the bottoms of the wells of interest may set to
the same height
prior to dispensing of fluid. Fluid may then be dispensed into the wells; it
will be appreciated
that in some cases, this may be done on-line, without moving the plate to
another location,
whereas in other cases, the plate may need to be moved to a dispensing
station. After dispensing
of the fluid, the auto-focus mechanism may again be employed (preceded if
necessary by return
of the plate to the auto-focus location), this time to determine the motion of
the well in the z-
axis; this information can in turn be used to determine the amount of fluid
dispensed in to one
or more wells, as described above. Moreover, periodic measurements may be
obtained to
determine if fluid has been lost from one or more wells, for example through
evaporation. This
method may be employed with individual wells or with groups
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wells, as described above. As noted, this method may be employed in
conjunction with or
instead of strain gauges to determine the displacement of one or more wells.
As with the methods already described above, detection of an incorrect amount
of
fluid in a well or group of wells facilitates correction of the amount of
fluid in the well(s),
exclusion of the well(s) from further manipulations and/or calculations, or in
some cases
correction of calculations.
Another method for determining the amount of fluid lost from a well involves
periodic monitoring of the temperature of the individual well. Often,
multiwell plates which
contain live cells are incubated at 37 C. However, the heat distribution in
the incubator may
be uneven, or other factors may cause uneven temperature distribution in the
plate, which can
lead to differential losses of fluid from different wells and can adversely
affect the cells
therein. By tracking the temperature of an individual well periodically, for
example once an
hour, and by taking into account the nature of the fluid in the well, it is
possible to determine
the amount of fluid lost from the well over time, as well as to correct the
temperature in the
well, for example using heating or cooling means as described hereinabove.
Such monitoring
may be facilitated by the placement of individual temperature sensors at each
well, for
example on the bottom or the side thereof. Such sensors may be electronically
coupled to a
card, such as 346 or 446 described above, to facilitate reading in a data
reader. Alternatively,
one or more thermal cameras may be employed to periodically detect the
temperature of
individual wells.
As with the methods already described above, detection of an incorrect amount
of
fluid in a well or group of wells facilitates correction of the amount of
fluid in the well(s),
exclusion of the well(s) from further manipulations and/or calculations, or in
some cases
correction of calculations. In cases in which the wells of the plate are
displaceable along the
z-axis, this method may be used in conjunction with or instead of strain
gauges, and in
conjunction with or instead of the method using an auto-focus mechanism as
described
above. However, it will be appreciated that unlike the methods using strain
gauges or auto-
focus, this method may be utilized with plates in which the wells are not
displaceable.
It will be appreciated that the embodiments shown in the figures are for
illustrative
purposes only and that variations of these are contemplated within the scope
of the invention.
For example, the number of wells per plate, the shape of the wells, and the
materials used
may differ what is shown or specifically described herein, as may the means
for detecting
adding or removal of liquid from the plate. Additionally, each of the wells
may include
additional layers or inserts, such as well inserts in which cells are grown
such that reagents
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can be added to the environment of the well for osmosis of the reagent into
the well without
directly engaging the cells grown in the insert.
It is appreciated that certain features of the invention, which are, for
clarity, described
in the context of separate embodiments, may also be provided in combination in
a single
embodiment. Conversely, various features of the invention, which are, for
brevity, described
in the context of a single embodiment, may also be provided separately or in
any suitable
subcombination or as suitable in any other described embodiment of the
invention. Certain
features described in the context of various embodiments are not to be
considered essential
features of those embodiments, unless the embodiment is inoperative without
those elements.
Although the invention has been described in conjunction with specific
embodiments
thereof, it is evident that many alternatives, modifications and variations
will be apparent to
those skilled in the art. Accordingly, it is intended to embrace all such
alternatives,
modifications and variations that fall within the scope of the appended
claims.
Citation or identification of any reference in this application shall not be
construed as
an admission that such reference is available as prior art to the invention.
Section headings are used herein to ease understanding of the specification
and should
not be construed as necessarily limiting.
43
