U.S. patent application number 15/233951 was filed with the patent office on 2016-12-01 for apparatus for measuring cell activity and method for analyzing cell activity.
The applicant listed for this patent is KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION. Invention is credited to Un-Hwan HA, Geon-Soo JIN, Seung-Pil PACK, Se-Hwan PAEK, Sung-Kyu SEO.
Application Number | 20160348058 15/233951 |
Document ID | / |
Family ID | 49632865 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160348058 |
Kind Code |
A1 |
SEO; Sung-Kyu ; et
al. |
December 1, 2016 |
APPARATUS FOR MEASURING CELL ACTIVITY AND METHOD FOR ANALYZING CELL
ACTIVITY
Abstract
The present invention relates to an apparatus that uses shadow
images of cells to continuously measure cell activity at a high
processing rate in order to provide cell activity and cell number
results. According to one embodiment of the present invention,
instead of a highly experienced examiner or technician using a
microscope, ELISA reader, etc. having to collect various cell
activity measurements and cell numbers, the collection of said
information can be automated so as to reduce cost and largely
reduce errors in measurements through the development of computer
software coupled with hardware using low cost and compact
optoelectronic components and simple image processing
techniques.
Inventors: |
SEO; Sung-Kyu; (Yongin-si,
KR) ; JIN; Geon-Soo; (Busan, KR) ; HA;
Un-Hwan; (Daejeon, KR) ; PAEK; Se-Hwan;
(Seoul, KR) ; PACK; Seung-Pil; (Daejeon,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION |
Seoul |
|
KR |
|
|
Family ID: |
49632865 |
Appl. No.: |
15/233951 |
Filed: |
August 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14385829 |
Sep 17, 2014 |
|
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PCT/KR2012/011555 |
Dec 27, 2012 |
|
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15233951 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 41/46 20130101;
G01J 1/0295 20130101 |
International
Class: |
C12M 1/34 20060101
C12M001/34; G01J 1/02 20060101 G01J001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2012 |
KR |
10-2012-0032808 |
Dec 3, 2012 |
KR |
10-2012-0138741 |
Claims
1. A cell activity measurement apparatus comprising: a cell chip
providing a cell storage place configured to receive a culture
fluid and a cell injected therein, the cell chip having a small
thickness of 0.1 mm-1.0 mm for a bottom surface thereof; a
light-emitting element positioned over the cell chip and configured
to emit light in a direction of the cell chip; and an image sensor
positioned under the cell chip and configured to capture a shadow
image of the cell.
2. The cell activity measurement apparatus of claim 1, further
comprising: a distance adjustment part coupled with the image
sensor and configured to adjust a distance between the image sensor
and the cell chip.
3. The cell activity measurement apparatus of claim 1, further
comprising: an analysis module configured to analyze an activity
state of the cell by using at least one of an SNR (signal-to-noise
ratio) value, an SD (shadow diameter) value, a maximum pixel value,
a position of a pixel having a maximum value, a minimum pixel
value, a position of a pixel having a minimum value, a diameter of
a first bright ring, a diameter of a first dark ring, a width
between a bright ring and a dark ring, an area of the shadow image,
and a circularity of the shadow image, extracted from the shadow
image captured by way of the image sensor.
4. The cell activity measurement apparatus of claim 3, wherein the
analysis module analyzes the activity state of the cell by using a
degree of similarity between a pixel for the shadow image captured
by way of the image sensor and a particular shape.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of U.S. Ser.
No. 14/385,829 filed Sep. 17, 2014, which claims the benefit of PCT
International Application No. PCT/KR2012/011555, which was filed on
Dec. 27, 2012, and which claims priority from Korean Patent
Application No. 10-2012-0032808, filed with the Korean Intellectual
Property Office on Mar. 30, 2012, and Korean Patent Application No.
10-2012-0138741, filed with the Korean Intellectual Property Office
on Dec. 3, 2012. The disclosures of the above patent applications
are incorporated herein by reference in their entirety.
BACKGROUND
[0002] 1. Technical Field
[0003] The present invention relates to an apparatus for measuring
cell activity and a method for analyzing cell activity, more
particularly to a cell activity measurement apparatus that uses
shadow images of cells to measure cell activity continuously and at
a high processing rate and a cell activity analysis method that can
analyze the measured cell activity.
[0004] 2. Description of the Related Art
[0005] Observing the activity of cells or whether the cells are
living or dead is an important step in developing modern medicines
or foods. The reactivity of cells to a particular drug or specimen
or observing whether the cells are living or dead is essential to
the research process. Some of the traditional techniques developed
for observing the proliferation or activity of cells are as
follows.
[0006] ELISA (enzyme-linked immunosorbent assay), which uses an
antibody having good specificity and high sensitivity together with
an enzyme serving as a signal source, is a technique that
selectively reacts cells that are in a particular state and
measures the absorbency.
[0007] Western blots refer to a method of detecting proteins by
electrophoresis. First, the proteins are separated by
polyacrylamide gel electrophoresis, and then the positions of the
electrophoresis separations are moved directly to a membrane, so as
to detect certain proteins by radioimmunoassay.
[0008] Immunohistochemistry is a method that uses a labeled
antibody to visualize certain antigens present in a tissue or a
cell, so that they may be observed through an optical microscope or
an electron microscope.
[0009] The methods listed above require large, expensive equipment
or entail dyeing cells and observing through a microscope or
measuring absorbency, etc., for the measuring of cell activity and
determining whether the cells are living or dead, and hence require
high costs, a complicated system, and a large space. Also, since
the specimen must inevitably be destroyed for analysis, it is
impossible to observe certain cells continuously.
[0010] Therefore, research is needed for a technology for measuring
cell activity that allows for quick and widespread use, without
requiring a separate reagent, and without destroying a
specimen.
SUMMARY
[0011] An objective of the present invention is to provide a cell
activity measurement apparatus based on shadow imaging technology
with which cells can be observed continuously without destroying
the specimen and without requiring a separate reagent
treatment.
[0012] An objective of the present invention is to provide a cell
activity analysis method that analyzes the activity of cells by
extracting certain parameters from a shadow image of the cells
captured via an image sensor.
[0013] To achieve the objectives above, an embodiment of the
present invention provides a cell activity measurement apparatus
that includes: a fluidic channel in which a culture fluid and a
cell are injected; a light-emitting element positioned over the
fluidic channel to emit light in a direction of the fluidic
channel; and an image sensor positioned under the fluidic channel
to capture a shadow image of the cell.
[0014] To achieve the objectives above, an embodiment of the
present invention provides a cell activity measurement apparatus
that includes: a well plate in which a culture fluid and a cell are
injected; a light-emitting element positioned over the well plate
to emit light in a direction of the well plate; and an image sensor
positioned under the well plate to capture a shadow image of the
cell.
[0015] To achieve the objectives above, an embodiment of the
present invention provides a cell activity measurement apparatus
that includes: a cell chip, which provides a cell storage place for
injecting a culture fluid and a cell, and which has a small
thickness of 0.1 mm-1.0 mm for its bottom surface; a light-emitting
element positioned over the cell chip to emit light in a direction
of the cell chip; and an image sensor positioned under the cell
chip to capture a shadow image of the cell.
[0016] To achieve the objectives above, an embodiment of the
present invention provides a cell activity measurement apparatus
that includes: a light-emitting element configured to emit light;
and an image sensor positioned under the light-emitting element to
receive a cell storage means placed thereon, where the cell storage
means includes a space in which a cell and a culture fluid are
injected, and the image sensor is configured to capture a shadow
image of the cell.
[0017] To achieve the objectives above, an embodiment of the
present invention provides a cell activity analysis method that
includes: receiving a shadow image captured by an image sensor;
calculating a particular parameter from the received shadow image;
and displaying an activity state of a cell by using the calculated
parameter.
[0018] A cell activity measurement apparatus according to an
embodiment of the present invention uses a simple, inexpensive
setup while allowing a continuous observation of a large amount of
cells without employing a separate reagent.
[0019] According to an embodiment of the invention, work that could
only be performed by experienced examiners or technicians who are
capable of using various cell activity measurement equipment such
as a microscope, etc., can be automated with the development of
computer software coupled with a simple image processing technique,
with the results of decreased costs and greatly reduced errors in
measurement.
[0020] Also, a cell activity analysis method according to an
embodiment of the invention may use pixel values from the shadow
image of cells, so that the activity of the cells can be easily
analyzed without expensive microscopes or devices such as ELISA
Reader.
[0021] Additional aspects and advantages of the present invention
will be set forth in part in the description which follows, and in
part will be obvious from the description, or may be learned by
practice of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a cell activity measurement apparatus related
to an embodiment of the present invention.
[0023] FIG. 2 illustrates the fluidic channel of FIG. 1 surrounded
by a temperature adjustment part.
[0024] FIG. 3 to FIG. 5 show a cell activity measurement apparatus
related to another embodiment of the present invention.
[0025] FIG. 6 is a block diagram of an analysis module related to
an embodiment of the present invention.
[0026] FIG. 7A, FIG. 7B, and FIG. 7c show a shadow image of cells
captured by way of the cell activity measurement apparatus of FIG.
1 and an image of the cells captured by way of a microscope.
[0027] FIG. 8 is a flow diagram illustrating a cell activity
analysis method related to an embodiment of the present
invention.
[0028] FIG. 9A and FIG. 9B illustrate a nucleus division state of a
cell as captured by way of the cell activity measurement apparatus
of FIG. 1.
[0029] FIG. 10A, FIG. 10B, and FIG. 10C illustrate a shadow image
captured by the cell activity measurement apparatus of FIG. 1 in
which live cells and dead cells are both present.
[0030] FIG. 11A, FIG. 11B, and FIG. 11C illustrate the shadow image
of FIG. 10A via an image processing technique.
[0031] FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D are diagrams for
describing a method of digitizing the number of cells in a cell
activity analysis method related to an embodiment of the present
invention.
DESCRIPTION OF NUMERALS
[0032] 100, 300, 400, 500: cell activity measurement apparatus
[0033] 110: light-emitting element [0034] 120: pinhole [0035] 130:
fluidic channel [0036] 131: flow cell [0037] 132: wall [0038] 133:
cover glass [0039] 140: image sensor [0040] 150: distance
adjustment part [0041] 160: analysis module [0042] 170: temperature
adjustment part
DETAILED DESCRIPTION
[0043] An apparatus for measuring cell activity and a method for
analyzing cell activity related to an embodiment of the present
invention will be described below in more detail with reference to
the accompanying drawings. The cell activity measurement apparatus
according to an embodiment of the invention can be implemented in
various ways according to the form of the cell storage means. The
cell storage means refers to a means that includes a space in which
cells are injected and cultivated or measured. For example, the
cell storage means can include a fluidic channel, a well plate, a
cell chip, etc.
[0044] In the present specification, an expression used in the
singular encompasses the expression of the plural, unless it has a
clearly different meaning in the context. In the present
specification, terms such as "comprising" or "including," etc.,
should not be interpreted as meaning that all of the components or
operations are necessarily included. That is, some of the
components or operations may not be included, while other
additional components or operations may be further included.
[0045] FIG. 1 shows a cell activity measurement apparatus related
to an embodiment of the present invention. The cell activity
measurement apparatus 100 here uses a fluidic channel as the cell
storage means.
[0046] As illustrated in the drawing, the cell activity measurement
apparatus 100 can include a light-emitting element 110, a pinhole
120, a fluidic channel 130, an image sensor 140, a distance
adjustment part 150, and an analysis module 160.
[0047] The light-emitting element 110 may emit light by which to
capture the shadow image of the cells. The light-emitting element
can be positioned at the fluidic channel 130 into which the cells
and a culture fluid (medium or media) may be injected. The
light-emitting element 110 can include an RGB light-emitting diode
(LED) for a clear capture of the shadow image.
[0048] The pinhole 120 can be coupled to a lower end of the
light-emitting element 110 to clarify the shadow image of the
cells. That is, the pinhole 120 can be used for increasing the
coherence and illuminance of the light.
[0049] The pinhole 120 can be fabricated in the form of a film mask
made of a plastic material. The film mask pinhole of a plastic
material can be printed on an OHP film, etc., with a
high-resolution laser printer and attached in front of the
light-emitting element 110, so that it is much more inexpensive to
fabricate compared with a pinhole of a metallic material and can be
fabricated easily. Also, in the case of a multi-wavelength light
source such as an RGB light-emitting diode where individual light
sources for three colors (red, green, and blue) are integrated in
one light-emitting diode, three pinholes may be positioned with a
gap of several tens of micrometers in-between, but by using a
method of printing with a high-resolution laser printer, the
multiple pinholes can be easily designed on a computer.
[0050] Inside the fluidic channel 130, a space may be prepared into
which the cells and the culture fluid may be injected and in which
the cells can be cultivated. The fluidic channel can be formed such
that the channel has a width of several mm or smaller, and thus can
reduce the volume of the culture fluid including the cells that is
injected therein. A reason for this is because a high concentration
of cells used for the activity measurement can result in great
noise during the analysis of the shadow image.
[0051] Also, at least a portion of the sidewall of the fluidic
channel 130 can be implemented with a polydimethylsiloxane (PDMS)
material. A reason for this implementation is that a supply of
oxygen may be required for the cultivation of cells, and the
inherent properties of the polydimethylsiloxane (PDMS) material
allow a permeation of oxygen. Thus, in cases where at least a
portion of the sidewall is implemented with a polydimethylsiloxane
(PDMS) material, a separate oxygen supply device may not be
required.
[0052] The fluidic channel 130 can include a flow cell 131, a wall
132, and a cover glass 133.
[0053] The flow cell 131 may be where the cells and the culture
fluid are injected and discharged and can serve as a cover for the
fluidic channel 130. The flow cell 131 can be fabricated using a
material such as glass, plastic, quartz, etc.
[0054] The wall 132 can be made of a polydimethylsiloxane (PDMS)
material and can be fabricated with a hollow middle portion so as
to provide a space between the flow cell 131 above and the cover
glass 133 below in which the cells may grow without permitting the
culture fluid to leak. In cases where the wall 132 is implemented
with a PDMS material, the properties of the PDMS material can
enable the permeation of oxygen, which is essentially required for
cell cultivation. Thus, it may not be necessary to provide the
fluidic channel 130 with a separate oxygen supply device. The wall
132 can be fabricated with a thickness of about 1 mm, to allow a
smaller size for the cell activity measurement apparatus 100.
[0055] The cover glass 133 can be positioned at the lower end of
the wall 132 and can serve as the bottom of the fluidic channel
130.
[0056] The image sensor 140 can capture the shadow image of the
cells at the lower portion of the fluidic channel 130. The image
sensor 140 can be implemented as a CMOS (complementary metal-oxide
semiconductor) image sensor. The CMOS image sensor is an
image-capturing element that has low power consumption and has a
complementary metal-oxide semiconductor (CMOS) structure. The image
sensor 140 can be implemented in a form that has no lens. The CMOS
image sensor has a fast processing speed and has a low cost because
it can be provided by mass production using semiconductor
processes. Also, it has a broader observing range compared to an
image sensor-based microscope using lenses and enables
quantification and automation of the analysis.
[0057] The distance adjustment part 150 can be coupled with the
image sensor 140 to adjust the distance between the image sensor
140 and the fluidic channel 130. That is, the distance adjustment
part 150 can serve to finely adjust the position of the image
sensor 140 to decrease the distance between the cover glass 133 and
the image sensor 140 as much as possible and capture the shadow
image with high contrast.
[0058] The analysis module 160 may serve to analyze the shadow
image captured with the image sensor 140 by way of an image
processing technique. The functions of the analysis module 160 will
be described later in more detail.
[0059] Also, the cell activity measurement apparatus 100 can
further include a temperature adjustment part for keeping the
cultivated cells at a constant temperature.
[0060] FIG. 2 illustrates the fluidic channel of FIG. 1 surrounded
by a temperature adjustment part.
[0061] As illustrated in the drawing, the temperature adjustment
part 170 can surround the fluidic channel 130 to serve as an
incubator such that the cells may be cultivated at a constant
temperature (e.g. 37.degree. C.). The temperature adjustment part
170 may be capable of temperature control and can include a
temperature sensor 171.
[0062] The shadow image of the cells captured by way of the image
sensor 140 can be transferred to the analysis module 160 where the
activity state of the cells may be analyzed.
[0063] FIG. 3 shows a cell activity measurement apparatus related
to an embodiment of the present invention. The cell activity
measurement apparatus 300 here uses a well plate as the cell
storage means.
[0064] As illustrated in the drawing, the cell activity measurement
apparatus 300 can include a light-emitting element 310, a pinhole
320, a well plate 330, an image sensor 340, a distance adjustment
part 350, and an analysis module 360.
[0065] The components illustrated in FIG. 1 can be applied in like
manner for the light-emitting element 310, pinhole 320, image
sensor 340, distance adjustment part 350, and analysis module 360,
and as such, the detailed descriptions of these components are
omitted here.
[0066] The well plate 330 may be positioned under the
light-emitting element 310 and may have a multiple number of wells
formed therein. A well refers to a space into which a specimen can
be injected.
[0067] For example, a 96-well plate or a 24-well plate can be used
for the well plate 330. A 96- or a 24-well plate refers to a well
plate in which 96 or 24 wells are formed.
[0068] In order to obtain a shadow image in which optical losses
are minimized, the well plate 330 can be a black well plate, which
has a thin well bottom and has little optical interference.
[0069] Also, the well plate 330 can be fabricated to be capable of
movement, to allow changes in the point at which the radiation from
the light-emitting element 310 arrives or changes in the point at
which the sensing by the CMOS image sensor 340 occurs. For example,
the well plate 330 can be fabricated to be rotatable.
[0070] Also, the cell activity measurement apparatus 300 can
further include a temperature adjustment part for keeping the
cultivated cells at a constant temperature.
[0071] FIG. 4 shows a cell activity measurement apparatus related
to an embodiment of the present invention. The cell activity
measurement apparatus 400 here uses a cell chip as the cell storage
means.
[0072] As illustrated in the drawing, the cell activity measurement
apparatus 400 can include a light-emitting element 410, a pinhole
420, a cell chip 430, an image sensor 440, a distance adjustment
part 450, and an analysis module 460.
[0073] The components illustrated in FIG. 1 can be applied in like
manner for the light-emitting element 410, pinhole 420, image
sensor 440, distance adjustment part 450, and analysis module 460,
and as such, the detailed descriptions of these components are
omitted here.
[0074] The cell chip 430 may be a cell storage means that is
implemented in the form of a chip that is provided with a space in
which to inject the cells and the culture fluid. The cell chip 430
may be a biochip that can detect the activity of cells or complex
physiological signals caused by cells and can be used for the
purposes of counting the cells.
[0075] A typical cell chip may be used with two optically
transparent sheets of glass, plastic, or polymer material, as an
upper substrate and a lower substrate, attached or assembled
together with a space in-between for storing the cells, and in the
case of a cell chip 430 for cell shadow imaging as used in an
embodiment of the present invention, a small thickness of about 0.1
mm-1.0 mm may be used for the thickness of the lower substrate.
Also, in the case of a cell chip that is fabricated in an
integrated form without attaching or assembling with a space
in-between for storing the cells, the thickness of the lower
substrate may be small, being about 0.1 mm-1.0 mm.
[0076] FIG. 5 shows a cell activity measurement apparatus related
to an embodiment of the present invention.
[0077] As illustrated in the drawing, cell activity measurement
apparatus 500 can include a light-emitting element 510, a pinhole
520, an image sensor 530, a distance adjustment part 540, and an
analysis module 550.
[0078] The components illustrated in FIG. 1 can be applied in like
manner for the light-emitting element 510, pinhole 520, image
sensor 530, distance adjustment part 540, and analysis module 550,
and as such, the detailed descriptions of these components are
omitted here.
[0079] However, the image sensor 530 can be implemented such that a
cell storage means can be placed thereon in a stable manner.
[0080] The cell activity measurement apparatus 500 does not include
the cell storage means as a component, but rather provides a space
in which to receive the cell storage means. That is, the cell
activity measurement apparatus 500 may be prepared with a space
where a cell storage means may be placed or received, and thus be
implemented to allow replacements of the cell storage means in
various forms as necessary, instead of being formed together with
the cell storage means as a set. For example, the cell activity
measurement apparatus 500 can have a well plate placed over the
image sensor 530 to measure the activity of the cells when a well
plate is required as the cell storage means, and can have a cell
chip placed over the image sensor 530 to measure the activity of
the cells when a cell chip is required as the cell storage
means.
[0081] Also, the cell activity measurement apparatus 500 can use a
particular cell storage means exclusively for placement or
reception. For example, the cell activity measurement apparatus 500
can be fabricated such that a cell chip is placed thereon
exclusively.
[0082] The cell activity measurement apparatuses 100, 300, 400, 500
described above can include the same analysis module.
[0083] FIG. 6 is a block diagram of the analysis module illustrated
in FIG. 1.
[0084] The illustrated analysis module 160 can be implemented as a
component of the cell activity measurement apparatus 100 but can
also be implemented as a device that is separate from the cell
activity measurement apparatus 100.
[0085] The analysis module 160 can include a receiver part 161, an
extractor part 162, a pixel filtering part 163, a display part 164,
and a control part 165.
[0086] The receiver part 161 can receive the shadow image from the
image sensor 140. In cases where the analysis module 160 exists
separately from the cell activity measurement apparatus 100, the
receiving of the shadow image can be performed by various
communication methods (e.g. wireless communication).
[0087] The extractor part 162 can calculate a particular parameter
for analyzing cell activity from the shadow image. For example, an
SNR (signal-to-noise ratio) value, an SD (shadow diameter) value,
the maximum pixel value, the position of a pixel having the maximum
value, the minimum pixel value, the position of a pixel having the
minimum value, the diameter of the first bright ring, the diameter
of the first dark ring, the width between a bright ring and a dark
ring, the area of the shadow image, the circularity of the shadow
image, and so on, can be a parameter for cell activity
analysis.
[0088] The SNR (signal-to-noise ratio) refers to a log value taken
after dividing the difference between the maximum intensity value
from pixels specified to include cells and the average intensity
value of the background that does not include cells by the standard
deviation value of the background.
[0089] The SNR can be expressed as Equation 1.
SNR=20 log|(max(I)-.mu..sub.b)/.sigma..sub.b|(dB) [Equation 1]
Here, .mu..sub.b and .sigma..sub.b refer to the average background
value of the shadow image and the standard deviation, respectively.
The value max(I) is the maximum intensity pixel value.
[0090] Also, the SD (shadow diameter) is a value expressing the
diameter of the cells' shadow image extracted from the intensity
values of pixels specified to include the cells as a root mean
square. The SD can be expressed as Equation 2.
S D = 2 x = 1 n ( x - x _ ) 2 f ( x ) 2 / x = 1 n f ( x ) 2 ( pixel
) [ Equation 2 ] ##EQU00001##
[0091] Here, n, x, and f(x) refer to the maximum number of pixels
in the cell shadow area, the specified pixels, and the intensity
values of the specified pixels, respectively.
[0092] The shadow image of the cells can be a circular image in
which bright rings and dark rings appear in an alternating manner.
In this case, the first bright ring from the center may be referred
to as a first bright ring, and the first dark ring from the center
may be referred to as a first dark ring.
[0093] Also, the circularity of a shadow image can be the measure
of how similar the shadow image is to a circle.
[0094] The pixel filtering part 163 can specify certain pixels from
the shadow image by using the particular parameter, similarity to a
particular shape, etc. This will be described later in more
detail.
[0095] The display part 164 can display the activity state of the
cells by using items extracted at the extractor part 162 for cell
activity analysis.
[0096] The control part 165 may control the overall functions
performed at the receiver part 161, extractor part 162, pixel
filtering part 163, and display part 164.
[0097] A method of analyzing the activity of cells is described
below with reference to test examples.
[0098] In the present test example, human aveolar epithelial cells
(A549) were used, which are epithelial cells of a human lung. The
initial concentration was 250,000 cells/ml. The oxygen demand of
the cells cultivated in the fluidic channel 130 was
5.795.times.10.sup.-7 mol/day, but the amount of oxygen permeation
at the wall 132 fabricated from a PDMS material was
1.771.times.10.sup.-6 mol/day, so that no separate oxygen supply
was necessary. A CO.sub.2 independent medium (18045, GIBCO), which
does not require a separate supply of CO.sub.2, was used for the
culture fluid that is injected for cell cultivation. The culture
fluid was injected into the fluidic channel 130 at a rate of 5-10
.mu.l/min by using a separate syringe pump (M200, KD Scientific).
In the present test example, an RGB light-emitting diode was used
for the light-emitting element 110, and a CMOS image sensor was
used for the image sensor 140.
[0099] FIGS. 7A to 7C shows a shadow image of cells captured by way
of the cell activity measurement apparatus of FIG. 1 and an image
of the cells captured by way of a microscope.
[0100] By using the cell activity measurement apparatus 100, the
nucleus division of a cell can be easily observed even without a
microscope. The cell of interest was observed through the cell
activity measurement apparatus 100 and a regular optical microscope
concurrently for 48 hours, and the comparison results show that the
nucleus division of the cell observed by the microscope
(photographs on the left) is also observable in the cell's shadow
image, as presented in FIG. 7A. By applying image processing
techniques on these changes in the shadow image, the pixel values
may be expressed as 3-dimensional (FIG. 7B) and 2-dimensional (FIG.
7C) graphs, from which it can be seen that rapid changes in the
shadow pattern occurred between 4 hours and 10 hours, when the
nucleus division occurred.
[0101] FIG. 8 is a flow diagram illustrating a cell activity
analysis method related to an embodiment of the present
invention.
[0102] The receiver part 161 can receive a shadow image of a cell
captured by way of the image sensor 140 (S810).
[0103] The extractor part 162 can calculate a particular parameter
from the cells' shadow image received (S820). In the test example,
the SNR value and the SD value were extracted from among the
parameters.
[0104] Then, the pixel filtering part 163 can specify particular
pixels from the shadow image by using a degree of similarity to a
particular shape (S830).
[0105] For example, the pixel filtering part 163 can choose and
specify only the pixels of which the circularity corresponds to a
particular range from among the pixels forming the shadow pattern
of the cell.
[0106] The display part 164 can draft and display time-lapse graphs
of the extracted SNR values and SD values (S840).
[0107] FIG. 9A and FIG. 9B show time-lapse graphs of the SNR values
and SD values extracted from the shadow image of a cell used in the
test example above.
[0108] As illustrated, the results of tracking the two items (SNR
and SD) for 48 hours are shown in FIG. 9A and FIG. 9B.
[0109] FIG. 9A represents changes in the SNR, and FIG. 9B
represents changes in the SD, and in both results, there are steep
changes between 4 hours and 10 hours from the beginning of the
test, when the nucleus division occurs.
[0110] FIG. 10A illustrates a shadow image captured by the cell
activity measurement apparatus of FIG. 1 in which live cells and
dead cells are both present.
[0111] For a comparative measurement of cell activity, cells were
cultivated in the cell activity measurement apparatus 100 for an
extended period, after which heat was applied to some cell colonies
to cause them to die. The test was performed such that one sheet of
the shadow image captured by the image sensor 140 includes
thousands of normal cell colonies (left) and dead cell colonies
(right), as in FIG. 10A. As a result, the living and dead cells
could be clearly differentiated under a microscope, as shown in
FIG. 10B and FIG. 10C.
[0112] As can be seen from FIGS. 10A to 10C, the cell activity
measurement apparatus 100 can observe cell activity to a greater
extent compared to the microscope.
[0113] FIGS. 11A to 11C illustrate the shadow image of FIG. 10A via
an image processing technique.
[0114] To quantify the live and dead cell colonies using the shadow
image, FIGS. 11A to 11C employ the SNR and SD values defined above
and show changes in these values for the living and dead cell
colonies.
[0115] As shown in FIG. 11A and FIG. 11B, between cells that are
living and cells that are dead, a greater magnitude of change in
the SNR and SD can be observed compared to what was observed during
the nucleus division of a cell. FIG. 11C shows how the shadow
pattern of a particular cell changes during a period of 60
hours.
[0116] FIGS. 12A to 12D are diagrams for describing a method of
digitizing the number of cells in a cell activity analysis method
related to an embodiment of the present invention.
[0117] By observing changes in the SNR and SD values of a shadow
pattern, it is possible to establish quantified criteria for
determining whether a cell is living or dead. FIG. 9A and FIG. 9B
show an example in which living and dead cells are quantified from
changes in the SNR and SD and in which this is used to count the
number of living cells. Also, an embodiment of the present
invention can include an operation of choosing certain pixels by
using the degrees of similarity between the pixels of the cell's
shadow image and a particular shape. From among the pixels forming
the shadow pattern of a cell, those pixels can be chosen and
specified of which the circularity corresponds to a particular
range. In this way, linearly shaped dust particles, etc., that are
differently shaped from the shadow pattern of a cell, which is
close to a circle, can be effectively identified and removed. The
measurement of the circularity of pixels can be calculated by
dividing the smaller of a shadow pattern's lateral length and
longitudinal length by the greater. A shadow image that is shaped
as a perfect circle would have a circularity value of 1, while a
lower circularity can have a value between 0 and 1.
[0118] FIG. 12A shows an example in which a preliminary evaluation
is performed regarding whether the cells are living or dead by
using SNR values extracted from the cells' shadow image (the area
above the dotted line in FIG. 11A represent living cells), and an
image processing technique is used to indicate red pixels. In the
preliminary evaluation, pixels having a particular SNR value
(approximately 18 dB) or higher were chosen and specified in red in
the overall shadow image.
[0119] FIG. 12B is an image showing a particular portion of FIG.
12A in a digitally magnified form. When a secondary evaluation is
performed regarding whether the cells are living or dead by using
SD values (the area below the dotted line in FIG. 11B represent
living cells), only the normal cells are shown in a silhouette
form, as illustrated in FIG. 12C.
[0120] In the secondary evaluation, the pixel clusters of which the
SD is smaller than or equal to a particular value (approximately 24
.mu.m) and at the same time the circularity of the pixels has a
value between 0.3-1.0 are selectively specified from among the red
pixel clusters selected from the preliminary evaluation.
[0121] Next, only the shadow images for cells that are thought to
be alive after the preliminary and secondary evaluation are
counted, for a numerical representation of the cells' activity.
[0122] FIG. 12D shows a digitization by an image processing
technique of the silhouette images for which the two-step
evaluation of cells using SNR and SD were completed. In this way,
the activity of the cells can be conclusively quantified. FIG. 12D
shows the results of counting the living cells remaining after
stopping the supply of culture fluid at T=36 hr, in order to
clearly present the quantification process of the cells'
activity.
[0123] As can also be seen from the test example described above, a
cell activity measurement apparatus 100 related to an embodiment of
the present invention can observe a large amount of cells
continuously without requiring a separate reagent. The cell
activity measurement apparatus 100 makes it possible to distinguish
a nucleus division in a cell or distinguish live and dead cells,
which in the past was observable only by means of an expensive
microscope, etc., using a separate dyeing reagent, etc.
[0124] In particular, a cell activity measurement apparatus
according to an embodiment of the present invention makes it
possible to perform cytotoxicity tests, which are essential in
developing new drugs, or perform measurements or evaluations of
microorganism activity in relation to environments and foods with
lost cost and in a speedy manner.
[0125] Moreover, work that could only be performed in the past by
experienced examiners or technicians who are capable of using
various cell activity measurement equipment such as a microscope,
etc., can be automated with the development of computer software
coupled with a simple image processing technique, with the results
of decreased costs and greatly reduced errors in measurement.
[0126] The cell activity analysis method described above can be
implemented in the form of program instructions that may be
performed using various computer means and can be recorded in a
computer-readable medium. Such a computer-readable medium can
include program instructions, data files, data structures, etc.,
alone or in combination. The program instructions recorded on the
medium can be designed and configured specifically for the present
invention or can be a type of medium known to and used by the
skilled person in the field of computer software.
[0127] Examples of a computer-readable medium may include magnetic
media such as hard disks, floppy disks, magnetic tapes, etc.,
optical media such as CD-ROM's, DVD's, etc., magneto-optical media
such as floptical disks, etc., and hardware devices such as ROM,
RAM, flash memory, etc.
[0128] Examples of a computer-readable medium can also include a
transmitting medium such as light, metal lines, waveguides, etc.,
that transmits signals for specifying program instructions, data
structures, etc.
[0129] Examples of the program of instructions may include not only
machine language codes produced by a compiler but also high-level
language codes that can be executed by a computer through the use
of an interpreter, etc. The hardware mentioned above can be made to
operate as one or more software modules that perform the actions of
the embodiments of the invention, and vice versa.
[0130] The cell activity measurement apparatus and cell activity
analysis method described above are not to be limited in their
application to the compositions and methods of the embodiments
described above. Rather, some or all of each of the embodiments may
be selectively combined to form numerous variations.
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