Patent Application
20200333202
The present invention relates to a force measurement method, a force measurement device, a force measurement system, a force measurement program, and a recording medium.
Priority is claimed on Japanese Patent Application No. 2017-210698, filed Oct. 31, 2017, the content of which is incorporated herein by reference.
Many eukaryotic cells have a developed intracellular transport system. In the related art, neurons have been used as a research subject for intracellular transport systems. Neurons have cell bodies and long extensions (axons) that extend from the cell body. The shape of a neuron is formed by a protein group constituting a cytoskeleton, and axons are mainly formed by tubulin constituting microtubules. Microtubules function as main lines of a logistics system (axonal transport) between the cell body and the synapse at the tip of the axon. Regarding examples of the cargo carried by axonal transport, intracellular organelles and vesicles covered by a lipid bilayer are known. Regarding examples of organelles, for example, mitochondria and endosomes are known. The vesicles contain substances that form axons and synapses, and proteins, nucleic acid polymers, and low-molecular-weight substances as information transmitting substances. Cargo transport includes anterograde transport from the cell body toward the tip of the axon and retrograde transport in a direction opposite thereto. In the anterograde transport, it is known that motor proteins called kinesin carry the cargo moving along microtubules. Similarly, in retrograde transport, motor proteins called dynein are responsible for transporting the cargo. Motor proteins transport the cargo using energy obtained by ATP hydrolysis.
Disfunction of axonal transport is thought to be one cause of neurological diseases such as Alzheimer's disease or Parkinson's disease, and analysis of axonal transport is expected to contribute to the understanding and treatment of neurological diseases.
While the cargo transport by motor proteins has been described above using axonal transport as a specific example, the transport of cargo by motor proteins is not limited to axonal transport. In neurons, the cargo is transported not only in the axon but also in extensions such as dendrites or in the cell body. In this case, the cargo is not necessarily covered with a lipid bilayer, but may be a protein complex or a protein-nucleic acid complex. In addition, regarding motor proteins, not only kinesin or dynein that move along microtubules, but also various motor molecules such as myosin that move on actin fibers may be used. In addition, intracellular transport is performed not only in neurons, but also in all eukaryotic cells according to the same mechanism. For example, in skin melanocytes, intracellular granules containing melanin are transported as cargo, and in insulin-secreting cells of the pancreas, intracellular vesicles containing insulin are transported as the cargo. That is, while the transport of cargo by motor proteins has been described above using neurons as a specific example, the transport of cargo by motor proteins is not necessarily performed only in neurons, and may also in cells other than neurons. For example, in cells other than neurons, the cargo transport by motor proteins is performed by motor proteins moving on microtubules in addition to the axon.
In the related art, in order to analyze such cargo transport, the force applied to cargo in living cells has been measured. A method of measuring a force applied to cargo in living cells is, for example, a method using optical tweezers. The method using optical tweezers is a method of applying a force to cargo to be observed using optical tweezers and measuring the force applied to the cargo by pulling the cargo. Here, the force applied to the cargo is, for example, a force applied to the cargo by motor proteins.
However, it is difficult to apply the method using optical tweezers other than to specific cargo such as oil droplets because a refractive index difference between the cargo and cytoplasm therearound, the size of the cargo, and an intensity of a laser beam determine a generated force. As a result, when the method using optical tweezers is applied to cargo other than oil droplets, a laser beam with an excessive intensity is necessary, and cell invasion and thermal damage may occur.
In this manner, since the method using optical tweezers is an invasive method using a laser beam, there is a problem that cells to be observed may be damaged.
In view of the above circumstances, an object of the present invention is to provide a technique for measuring a force applied to cargo in living cells in a non-invasive manner.
One aspect of the present invention is a force measurement method including a first acquisition step in which, among data included in time course data indicating a movement trajectory of cargo acquired in a non-invasive manner, first data indicating a temporal change of a position of the cargo during a period in which the displacement of the cargo per unit time is substantially constant is acquired, a fluctuation value calculation step in which, based on a first probability distribution indicating a distribution of a probability that the amount of change at a predetermined position of the cargo per unit time is a predetermined amount, which is a probability distribution based on the first data, a fluctuation value proportional to a force applied to the cargo by motor proteins that transport the cargo is calculated, and a force calculation step in which the force is calculated based on the calculated fluctuation value.
In another aspect of the present invention, in the force measurement method, in the force calculation step, the force is calculated by performing clustering on the fluctuation value.
In another aspect of the present invention, the force measurement method further includes a second acquisition step in which, among time course data indicating a movement trajectory of the cargo acquired in a non-invasive manner, time course data including the first data and second data indicating a temporal change of the position of the cargo during a period in which the position of the cargo is substantially the same position regardless of time is acquired; and a fluctuation coefficient calculation step in which a fluctuation coefficient which is a conversion coefficient with respect to between the force and the fluctuation value is calculated based on the first and second data.
In another aspect of the present invention, in accordance with the force measurement method, in the fluctuation coefficient calculation step, a viscosity coefficient of a medium surrounding the cargo is calculated based on the second data.
In another aspect of the present invention, in accordance with the force measurement method, in the fluctuation coefficient calculation step, a speed at which the cargo moves is calculated based on the first data acquired in the second acquisition step.
In another aspect of the present invention, in accordance with the force measurement method, in the fluctuation coefficient calculation step, the fluctuation coefficient is calculated based on the viscosity coefficient and the speed.
In another aspect of the present invention, in accordance with the force measurement method, in the fluctuation value calculation step, χ is calculated according to a formula of χ=ln[P1(ΔX1)/P1(−ΔX1)]ΔX1 when the amount of change in the position is set as ΔX, a probability that the value of ΔX during the unit time is ΔX1 is set as a first probability P1(ΔX1), and the fluctuation value is set as χ.
Another aspect of the present invention is a force measurement device, including: a first acquisition unit configured to acquire first data indicating a temporal change of a position of cargo during a period in which the displacement of the cargo per unit time is substantially constant among data included in time course data indicating a movement trajectory of the cargo acquired in a non-invasive manner, a fluctuation value calculation unit configured to calculate, based on a first probability distribution indicating a distribution of a probability that the amount of change at a predetermined position of the cargo per unit time is a predetermined amount, which is a probability distribution based on the first data, a fluctuation value proportional to a force applied to the cargo by motor proteins that transport the cargo; and a force calculation unit configured to calculate the force based on the calculated fluctuation value.
Another aspect of the present invention is a force measurement system, including: a first acquisition unit configured to acquire first data indicating a temporal change of a position of cargo during a period in which the displacement of the cargo per unit time is substantially constant among data included in time course data indicating a movement trajectory of the cargo acquired in a non-invasive manner, a fluctuation value calculation unit configured to calculate, based on a first probability distribution indicating a distribution of a probability that the amount of change at a predetermined position of the cargo per unit time is a predetermined amount, which is a probability distribution based on the first data, a fluctuation value proportional to a force applied to the cargo by motor proteins that transport the cargo; and a force calculation unit configured to calculate the force based on the calculated fluctuation value.
Another aspect of the present invention is a force measurement program causing a computer to acquire first data indicating a temporal change of a position of cargo during a period in which the displacement of the cargo per unit time is substantially constant among data included in time course data indicating a movement trajectory of the cargo acquired in a non-invasive manner; to calculate, based on a first probability distribution indicating a distribution of a probability that the amount of change at a predetermined position of the cargo during unit time is a predetermined amount, which is a probability distribution based on the time course data, a fluctuation value proportional to a force applied to the cargo by motor proteins that transport the cargo; and calculate the force based on the calculated fluctuation value.
Another aspect of the present invention is a recording medium storing a program causing a computer: to acquire first data indicating a temporal change of a position of cargo during a period in which the displacement of the cargo per unit time is substantially constant among data included in time course data indicating a movement trajectory of the cargo acquired in a non-invasive manner; to calculate, based on a first probability distribution indicating a distribution of a probability that the amount of change at a predetermined position of the cargo during unit time is a predetermined amount, which is a probability distribution based on the time course data, a fluctuation value proportional to a force applied to the cargo by motor proteins that transport the cargo; and calculate the force based on the calculated fluctuation value.
According to the present invention, a force applied to cargo in living cells can be measured in a non-invasive manner.
In the present invention, when a physical quantity related to the force applied to cargo by the fluctuation theorem is measured, the force applied to the cargo is measured. Therefore, in description of the present invention, first, the fluctuation theorem will be described, and a principle in which a force applied to cargo is measured according to the present invention will be then described.
The fluctuation theorem is a theorem of nonequilibrium statistical mechanics, a field of physics, published in 1993, D. J. Evans et al. The fluctuation theorem is a theorem that links a predetermined physical quantity in a thermodynamically nonequilibrium system to a physical quantity that changes the predetermined physical quantity. The thermodynamically nonequilibrium system is, for example, a system in which cargo moves. In this case, the fluctuation theorem links the position of the cargo in the system to the force applied to the cargo. In this case, it is known that the fluctuation theorem that links the position of the cargo that moves and the force applied to the cargo that moves is represented by the following formula.
ΔX indicates an amount of displacement of the cargo per unit time, and P(ΔX) indicates a probability that an amount of displacement of the cargo is ΔX. Here, ΔX is represented by the following Formula (2).
[Math. 2]
ΔX=X(t+Δt)−X(t) (2)
In Formula (2), X(t) is a position of the cargo at the time t. In Formula (2), X(t+Δt) is a position of the cargo at the time t+Δt.
In ΔX, a force Fc (denoted as F in Formula (1)) indicates a magnitude of a force applied to the cargo. A is a conversion coefficient for the physical quantity represented by the left side in Formula (1) and the force Fc. Hereinafter, the physical quantity represented by the left side in Formula (1) is a fluctuation value χ, and A is a fluctuation coefficient. The fluctuation coefficient A is a unique value in a combination of the cargo and motor proteins.
When the amount of displacement of the cargo is measured, the fluctuation value χ is calculated. Therefore, when the fluctuation coefficient A is provided, the force Fc applied to the cargo can be calculated by Formula (1).
Here, the amount of displacement of the cargo indicates the amount of change in the position of the cargo. In addition, the position of the cargo may be any position as long as it is a position of a part of the cargo, and may be, for example, the position of the center of gravity of the cargo.
The probability distributions in
Therefore, the height of the peak of the probability distribution in
When the displacement of the cargo is measured, not only the value on the left side in Formula (1) but also the fluctuation coefficient A can be calculated. This will be described below. In cargo that moves in a viscous medium at a constant speed V, the force applied to the cargo by motor proteins and the viscous resistance force due to the medium match. Therefore, if the viscosity coefficient F of the medium is known, the force Fc applied to the cargo by motor proteins can be calculated. Specifically, the force Fc and the viscosity coefficient r satisfy the relationship of the following Formula (3).
[Math. 3]
F
c
=ΓV (3)
Therefore, based on the fluctuation value χ and the force Fc calculated based on the viscosity coefficient Γ, the fluctuation coefficient A is calculated. Here, the fluctuation coefficient A is a product of a Boltzmann constant kb, and a temperature Teff (that is, the temperature is generally referred to as a nonequilibrium temperature or an effective temperature) of a system including cargo to be observed in the nonequilibrium state. The nonequilibrium state is a state in which the cargo is being transported.
In transport of the cargo according to motor proteins, Teff is a constant value. This has been experimentally verified.
Here, in the present invention, the cell to be observed has cargo and motor proteins and may be any cell as long as the motor proteins in the cell transport the cargo in the cell. Many generally known eukaryotic cells have such cargo and motor proteins. The observation subject is preferably neurons because in these the transport of cargo in the cell is easily observed. Examples of cells suitable as the observation subject other than neurons include glial cells, vascular endothelial cells, fibroblasts of skin or the like, melanocytes, generally used established animal cells, and egg cells.
In the present invention, the type of neurons to be observed is not particularly limited, and for example, neurons that can be cultured in vitro are preferable because it is easy to capture a video in these neurons. In addition, they may be cells that support neurons or cells in which other cells coexist. Examples of such cells include glial cells such as astrocytes, oligodendrocyte, and Schwann cells, vascular cells such as vascular epithelial cells, and mesenchymal cells such as fibroblasts. In addition, examples of such neurons include neurons that secrete an amino acid type neurotransmitter, neurons that secrete a peptide type neurotransmitter, and neurons that secrete monoamines or acetylcholine. When neurons are cultured, cells that support neurons may be allowed to coexist in order to simulate an in vivo environment. Examples of such accessory cells include glial cells such as astrocytes, oligodendrocyte, and Schwann cells.
In addition, in the present invention, the type of the cargo transported in cells to be observed is not particularly limited and may be any cargo of which fluctuation can be measured by an optical device. The presence or absence of a fluorescent label of the cargo when the fluctuation is measured is not limited. When the cargo is a vesicle or organelle covered with a lipid bilayer, it can be fluorescently labeled with a carbocyanine dye, for example, DiD, DiI, DiO, or DiR. Alternatively, proteins contained in the cargo can be fluorescently labeled with a fluorescent protein such as GFP. In addition, staining using a fluorescent dye and known labeling methods using biotin/avidin, antibodies, and the like can also be applied. The transport path of the cargo to be observed is not particularly limited, and may be axonal transport or other intracellular transports and can be appropriately selected according to the purpose of analysis.
The force measurement device 1 calculates a force Fe applied to the cargo to be observed based on a data group of images showing movement of the cargo to be observed. Hereinafter, a data group of images showing movement of the cargo to be observed will be referred to as observation data. The image showing movement of the cargo to be observed may be, for example, a 2D image. In addition, the observation data may be, for example, a video.
The fluctuation coefficient calculation device 2 calculates the fluctuation coefficient A of the cargo to be observed based on the observation data. The fluctuation coefficient calculation device 2 outputs the calculated fluctuation coefficient A to the force measurement device 1.
The imaging device 9 images the cargo to be observed in a non-invasive manner, and generates observation data in which movement of the cargo is recorded. The non-invasive manner may be, for example, a manner in which information on an observation subject is acquired with visible light. The information on an observation subject is, for example, a signal indicating the position of the center of gravity of the observation subject. In addition, the non-invasive manner may be, for example, a manner in which an observation subject is labeled with fluorescence to acquire information on an observation subject based on the fluorescence. These non-invasive manners may be realized using a microscope such as a fluorescence microscope or a differential interference microscope.
In addition, the non-invasive manner may be a manner in which information on an observation subject is acquired using a magnetic field with an intensity having little influence on the observation subject. Here, the invasive manner is, for example, a manner in which information on an observation subject is acquired by emitting a laser beam to an observation subject such as optical tweezers.
Here, a method of generating observation data in which movement of the cargo is recorded is not particularly limited, and for example, a known method using a confocal laser microscope including a commercially available digital video camera for cell dynamics observation may be used. In consideration of the speed of cargo axonal transport, and the like, an imaging frame rate is preferably about 10 to 100 frames/sec or more and particularly preferably 100 frames/sec.
In addition, in a process or manner of extracting an axon region in the captured image frame, various methods can be applied. For example, a method of visually determining an axon region is performed.
In
Here, the cargo to be observed is cargo to be observed in the region 101 in
The observation data is a data group including a plurality of 2D stationary images shown in
The first trajectory data acquisition unit 11 acquires time course data including constant speed movement data generated based on observation data. The time course data is data indicating projection of the observation subject indicated by observation data on one predetermined axis of a trajectory and is a set of data having two values of a time t and a position X of the cargo at the time t. The one predetermined axis may be any axis, and may be, for example, an axis parallel to a direction in which the cargo is transported. In addition, the constant speed movement data is data included in time course data and is data indicating a temporal change of the position of the cargo during a period in which the displacement of the cargo per unit time is substantially constant (hereinafter referred to as a “constant speed period”). That is, the constant speed movement data is data indicating a temporal change of the position of the cargo during a period in which the cargo moves at a substantially constant speed. The constant speed movement data is also a set of data having two values of the time t and the position X of the cargo.
The graph in
The description will return to
The first probability distribution calculation process is a process of calculating a probability distribution of the amount of displacement ΔX of the position of the cargo in the unit time Δt during a constant speed period based on one piece of constant speed movement data. Specifically, in the first probability distribution calculation process, first, the amount of displacement ΔXn=Xn−X(n-1) of the position of the cargo is acquired for various n when the time origin in constant speed movement data is t0, the position of the cargo at the time origin t0 is X0, and the position of the cargo at the time t0+n×Δt (n is an integer) is Xn. Hereinafter, a process of acquiring the amount of displacement ΔXn of the position of the cargo for various n is referred to as a displacement amount calculation process. Next, in the first probability distribution calculation process, a probability distribution (hereinafter referred to as a “first probability distribution”) in which the value of the amount of displacement ΔX of the position of the cargo is ΔXn is calculated.
The graph in
The description will return to
The repetitive control unit 13 controls the fluctuation value calculation unit 12 so that a plurality of fluctuation values χ are calculated. Specifically, first, the repetitive control unit 13 controls the fluctuation value calculation unit 12 so that the a fluctuation value χ is calculated at a plurality of predetermined unit times Δt from the shortest unit time to the longest unit time with respect to one piece of constant speed movement data. Hereinafter, this control is referred to as unit temporal change repetitive control. The longest unit time is a predetermined time that is determined in advance by a user and is the shortest time among possible times of the unit time Δt. The longest unit time is a predetermined time that is determined in advance by a user and the longest time among possible times of the unit time Δt. The shortest unit time may be, for example, 10 ms. The longest unit time may be, for example, 100 ms. In addition, the unit time may be, for example, 50 ms. In addition, the repetitive control unit 13 performs unit temporal change repetitive control on the time course data including constant speed movement data acquired by the first trajectory data acquisition unit 11.
Here, the unit temporal change repetitive control may be, for example, a control in which the fluctuation value calculation unit 12 is controlled so that the fluctuation value χ is calculated at each Δt in order from the shorter unit time Δt from the shortest unit time to the longest unit time.
The description will return to
Next, the clustering unit 14 outputs the representative value of the result of clustering as χ in which the variation in measurement is reduced. Here, the representative value may be any value as long as the value represents a cluster for each clustering method, and may be, for example, an average value or a median value.
The fluctuation coefficient storage unit 15 stores the fluctuation coefficient A calculated by the fluctuation coefficient calculation device 2.
The fluctuation coefficient storage unit 15 is configured using a storage device such as a magnetic hard disk drive and a semiconductor storage device.
The force calculation unit 16 calculates a force Fe applied to the cargo to be observed based on the fluctuation value χ and the fluctuation coefficient A. Specifically, Fc is calculated using Formula (1) and the fact that the right side of Formula (1) is χ.
The second trajectory data acquisition unit 21 acquires time course data including constant speed movement data and stationary data generated based on observation data. The stationary data is data included in the time course and is data indicating a temporal change in the position of the cargo during a period in which the position of the cargo is substantially the same position regardless of time (hereinafter referred to as “stationary period”). That is, the stationary data is data indicating a temporal change of the position of the cargo during a period in which the cargo remains at the same position regardless of time. Like the time course data, the stationary data is a set of data having two values of the time t and the position X of the cargo. Here, among the time course data including constant speed movement data, a proportion of time course data including not only constant speed movement data but also stationary data is about 10%.
The graph in
The description will return to
The second probability distribution calculation process is a process of calculating a probability distribution (hereinafter referred to as a “second probability distribution”) of the position X of the cargo at the time t during the stationary period based on one piece of stationary data. The second probability distribution is a distribution according to the Boltzmann distribution.
In the distribution coefficient calculation process, fitting is performed on the second probability distribution calculated in the second probability distribution calculation process using a Boltzmann function PB(X), which is a function including K as a fitting parameter, and the value of the fitting parameter K is calculated. A specific function form of the Boltzmann function PB(X) is a function form represented by Formula (4). Hereinafter, the fitting parameter K will be referred to as a distribution coefficient. Here, the fitting may be performed by any fitting method, for example, a least squares method.
PB(X) represents a probability that the position of the cargo is X. Z and T represent a distribution function and the temperature of an environment. The temperature of the environment is a temperature of a medium surrounding the cargo to be observed.
The power spectrum calculation process is a process of Fourier-transforming the second probability distribution according to the frequency ν and calculating a power spectrum.
The power coefficient calculation process is a process in which the power spectrum is fitted using a Lorentzian function g(ν) including Ap and Bp as fitting parameters and a fitting parameter Bp is calculated. A specific function form of the function g(ν) is a function form represented by Formula (5). Hereinafter, the fitting parameter Bp will be referred to as a power coefficient. The fitting may be performed by any fitting method, for example, a least squares method.
The viscosity coefficient calculation process is a process of calculating a viscosity coefficient Γ represented by the following Formula (6) using the distribution coefficient K and the power coefficient Bp.
[Math. 6]
Γ=KBp (6)
The fluctuation coefficient calculation unit 23 calculates the fluctuation coefficient A based on the viscosity coefficient Γ and the fluctuation value χ. The viscosity coefficient calculation unit calculates a fluctuation coefficient A by sequentially performing a speed calculation process and a fluctuation coefficient calculation process.
The speed calculation process is a process of calculating a time average speed V of the cargo during a constant speed movement period using constant speed movement data of time course data including stationary data. Specifically, the speed calculation process is a process of calculating a slope of a regression line of constant speed movement data in a graph in which the horizontal axis represents time and the vertical axis represents the position of the cargo.
The fluctuation coefficient calculation process is a process of calculating a fluctuation coefficient A based on the speed V, the viscosity coefficient Γ and the fluctuation value χ. In the fluctuation coefficient calculation process, first, the fluctuation coefficient calculation unit 23 acquires data having two values of the force Fc calculated by Formula (3) and the fluctuation value χ calculated by the fluctuation value calculation unit 12 (hereinafter referred to as “force fluctuation value correspondence data”) for a plurality of pieces of time course data including stationary data and constant speed movement data. Next, in the fluctuation coefficient calculation process, the fluctuation coefficient calculation unit 23 fits the acquired plurality of pieces of force fluctuation value correspondence data using a linear function represented by the following Formula (7) including a fitting parameter A1.
[Math. 7]
F
c
=A
1χ (7)
A1 is a fitting parameter, and the value of A1 obtained by fitting is a fluctuation coefficient A. It is clear from Formula (1) and Formula (6) that the value of A1 obtained by fitting is the fluctuation coefficient A.
The imaging device 9 generates observation data of the cargo to be observed (Step S101). The first trajectory data acquisition unit 11 acquires time course data including constant speed movement data generated based on observation data acquired by the imaging device 9 (Step S102). The repetitive control unit 13 performs unit time repetitive control on one piece (hereinafter referred to as “data to be calculated”) of time course data including constant speed movement data at a plurality of predetermined unit times Δt and thus calculates the fluctuation value χ at the plurality of unit times Δt (Step S103).
The repetitive control unit 13 determines whether there is time course data on which the first probability distribution calculation process has not been performed among time course data including constant speed movement data acquired by the first trajectory data acquisition unit 11 (Step S104). When there is no time course data on which the first probability distribution calculation process has not been performed (No in Step S104), the clustering unit 14 performs clustering on the fluctuation value χ and calculates a fluctuation value χ in which the variation in measurement is reduced (Step S105).
After the clustering unit 14 calculates the fluctuation value χ in which the variation related to measurement is reduced, the second trajectory data acquisition unit 21 acquires time course data including stationary data and constant speed movement data (Step S106). After the second trajectory data acquisition unit 21 acquires time course data including stationary data and constant speed movement data, the viscosity coefficient calculation unit 22 performs the second probability distribution calculation process, and thus calculates a second probability distribution based on the stationary data acquired by the second trajectory data acquisition unit 21 (Step S107). After the second probability distribution is calculated, the viscosity coefficient calculation unit 22 performs the distribution coefficient calculation process on the second probability distribution and calculates the value of the distribution coefficient K in the Boltzmann function PB(X) represented by Formula (4) (Step S108). The viscosity coefficient calculation unit 22 that has calculated the distribution coefficient K performs the power spectrum calculation process on the second probability distribution and calculates a power spectrum (Step S109). The viscosity coefficient calculation unit 22 that has calculated the power spectrum performs the viscosity coefficient calculation process and calculates a viscosity coefficient Γ (Step S110).
After the viscosity coefficient calculation unit 22 calculates a viscosity coefficient Γ, the fluctuation coefficient calculation unit 23 performs the fluctuation coefficient calculation process and calculates a fluctuation coefficient A (Step S111). After the fluctuation coefficient A is calculated, the force calculation unit 16 calculates a force Fc applied to the cargo based on χ calculated in Step S105 and the fluctuation coefficient A calculated in Step S111 (Step S112).
On the other hand, in Step S104, when there is time course data on which the first probability distribution calculation process has not been performed (Yes in Step S104), the repetitive control unit 13 sets the time course data as new data to be calculated (Step S113). The repetitive control unit 13 performs unit time repetitive control on new data to be calculated (Step S103).
Here, Steps S106 to 110 need not necessarily be performed after Step 107, and may be performed in parallel with Steps S103 to 105, or may be performed before Steps S103 to 105.
Since the force measurement device 1 of the embodiment configured in this manner includes the fluctuation value calculation unit 12 that calculates the value χ proportional to the force Fc applied to the cargo to be observed based on a 2D image data group indicating movement of the cargo, the user can measure the force applied to cargo in living cells in a non-invasive manner.
The superior cervical ganglion was collected from 3-week old ICR mice (male) and treated with an enzyme such as 0.5% trypsin and 0.5% collagenase. neurons separated individually were rinsed with a DMEM/F12 medium containing a 10% h inactivated bovine serum, and then spread on a matrigel-coated glass bottom dish. The neurons were cultured in a DMEM/F12 medium containing a 10% inactivated bovine serum and a 200 ng/ml 2.5 S nerve growth factor in an incubator at 37° C. and CO2 5% for 2 to 4 days and used for an experiment using the force measurement device 1.
A fluorescent dye DiI was added to a medium of neurons during culturing so that the final concentration was 100 nM, and staining was performed for 10 minutes, and the result was then used for fluorescence observation.
The dish after stain was placed on a heating plate (CU-201, Live Cell Instrument), and the stained melanin vesicles in melanocytes were observed under a dark-field microscope (IX71, Olympus) while the temperature was maintained at 37° C. A video was captured using an EMCCD camera (LucaS (Andor)) with a 100× objective lens (UPlanFL 100x/1.3, Plympus) at 98 frames per second. Time course data of the position of the center of gravity of melanin vesicles was calculated from the video using ImageJ (Rasband, 1997).
Comparing
Comparing
Functional units of the force measurement device 1 and the fluctuation coefficient calculation device 2 need not necessarily be mounted in different housings but may be mounted in one housing.
Here, the second trajectory data acquisition unit 21 need not necessarily acquire time course data including constant speed movement data and stationary data based on observation data, but may acquire time course data acquired by the first trajectory data acquisition unit 11 and acquire time course data including constant speed movement data and stationary data among the acquired time course data.
Here, the time course data including constant speed movement data acquired by the first trajectory data acquisition unit 11 may be generated by a user based on observation data or may be generated by a device. In addition, the time course data including constant speed movement data and stationary data acquired by the second trajectory data acquisition unit 21 may be generated by a user based on observation data or may be generated by a device. The constant speed movement data used by the fluctuation value calculation unit 12 to calculate a fluctuation value may be selected manually by a user or selected by a device.
The constant speed movement data and stationary data used by the fluctuation coefficient calculation device 2 may be selected manually by a user or may be selected by a device. In addition, the cargo to be observed may be selected manually by a user or may be selected by a device.
Here, the image data group and observation data need not necessarily be 2D data but may be 3D or 1D data.
The force measurement device 1 and the fluctuation coefficient calculation device 2 in the above-described embodiments may be realized in a computer. In this case, a program for realizing this function is recorded in a computer readable recording medium, and a computer system is caused to read and execute the program recorded in the recording medium for realization. Here, “computer system” includes an OS and hardware such as peripheral devices. In addition, “computer readable recording medium” refers to a portable medium such as a flexible disk, a magneto-optical disc, a ROM, and a CD-ROM, and a storage device such as a hard disk built into a computer system. In addition, the “computer readable recording medium” may include a medium that dynamically maintains a program for a short time like a communication line when a program is transmitted via a network such as the Internet or a communication line such as a telephone line, and a medium that maintains a program for a certain time like a volatile memory in the computer system serving as a server or a client in that case. In addition, the program may be a program for realizing some of the above-described functions, a program that can be realized in combination with a program in which the above-described functions are already recorded in a computer system, or a program that is realized using a programmable logic device such as a field programmable gate array (FPGA).
In the above-described embodiments, the present invention is expressed as the force measurement device, but the present invention can also be expressed as a force measurement method. In this case, the force measurement method according to the present invention corresponding to the force measurement device in the embodiment can be expressed as a force measurement method including a first acquisition step in which, among data included in time course data indicating a movement trajectory of cargo acquired in a non-invasive manner, first data indicating a temporal change of a position of the cargo during a period in which the displacement of the cargo per unit time is substantially constant is acquired, a fluctuation value calculation step in which, based on a first probability distribution indicating a distribution of a probability that the amount of change at a predetermined position of the cargo per unit time is a predetermined amount, which is a probability distribution based on the first data, a fluctuation value proportional to a force applied to the cargo by motor proteins that transport the cargo is calculated, and a force calculation step in which the force is calculated based on the calculated fluctuation value.
In the force measurement method corresponding to the force measurement device in the above embodiment, the first acquisition step corresponds to an operation or process of the first trajectory data acquisition unit 11 in the embodiment, and the fluctuation value calculation step corresponds to an operation or process of the fluctuation value calculation unit 12 and the repetitive control unit 13 in the embodiment. In addition, the force calculation step corresponds to an operation or process of the force calculation unit 16 in the embodiment.
Here, the operation or process of the second trajectory data acquisition unit 21 in the embodiment is an example of the second acquisition step, and the operation or process of the fluctuation coefficient calculation unit 23 in the embodiment is an example of the fluctuation coefficient calculation step.
Here, the first trajectory data acquisition unit 11 is an example of a first acquisition unit. Here, the second trajectory data acquisition unit 21 is an example of a second acquisition unit. Here, constant speed movement data is an example of first data. Here, stationary data is an example of second data.
While the embodiments of the invention have been described above in detail with reference to the drawings, specific configurations are not limited to the embodiments, and include designs and the like without departing from the spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-210698 | Oct 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/040300 | 10/30/2018 | WO | 00 |
