JP5945434B2 - Biological sample image analysis method, image analysis apparatus, image capturing apparatus, and program - Google Patents

Description

  The present invention relates to a biological sample image analysis method, an image analysis apparatus, an image capturing apparatus, and a program.

[Gene expression]
The cell transcribes an appropriate portion as RNA from DNA as genetic information at an appropriate time. Most of the transcribed RNA is mRNA, which is translated into protein, functions as a cell structure and an enzyme, and supports cell activities. Some RNAs are not translated into proteins, but play an important role in maintaining cellular functions. Thus, the production of a protein or RNA having a function from DNA is called gene expression.

Directly responsible for gene expression is RNA polymerase responsible for RNA transcription. RNA polymerase recognizes and binds to a sequence called a promoter region on DNA, and starts transcription of RNA from a position specified by the promoter.
A group of proteins called transcription factors binds to a unique promoter region and its surrounding region (enhancer region) on the DNA sequence to promote or inhibit the binding of RNA polymerase. By doing so, the transcription factor plays a role in regulating the amount of expression of the corresponding gene.

  Therefore, the expression of a gene encoding a transcription factor controls the expression of another gene. Some transcription factors recognize and bind to a plurality of regions on genomic DNA. In many cases, gene expression forms a complex network involving many genes via these transcription factors.

  There are various beginnings of gene expression, one of which is signal transduction from outside the cell. Signal transduction is initiated by the binding of a ligand to a receptor on the cell membrane. Downstream of the signal transduction pathway, a gene expression network corresponding to each signal is induced via a plurality of transcription factors. This mechanism allows cells to respond to the outside world.

Another example of a gene expression network is a clock gene network. Clock genes are a group of genes that control the circadian rhythm (circadian rhythm) of an organism.
The circadian rhythm exists in almost all living organisms regardless of whether they are unicellular organisms or multicellular organisms. In eukaryotes, the circadian rhythm of individual cells is realized by a feedback loop of a gene expression network via clock genes.

  In the case of multicellular organisms, the circadian rhythm of individual cells can be engraved with the same rhythm as a whole by synchronizing between cells via the signal transduction system described above. In particular, in mammals, there is a clock center in the suprachiasmatic nucleus in the hypothalamus of the brain, and synchronizes the circadian rhythm of the individual.

Gene expression networks are necessary for cell maintenance, growth, and differentiation. For molecular and cell biology, elucidating gene expression networks related to individual phenomena is one of the major research topics. is there.
[Biochemical and immunological gene expression analysis methods]
A conventional method for analyzing gene expression is a method of detecting a gene expression product biochemically or immunochemically and analyzing the result. For example, when the gene expression product is a protein, gene expression can be detected with an antibody specific to the protein. When the gene expression product is RNA, gene expression can be detected by hybridization or reverse transcription polymerase chain reaction.

These detection methods are used for biochemical methods by homogenizing all or part of a biological sample, and also used for imaging the expression distribution of immobilized samples by immunostaining or in situ hybridization. ing.
However, in these methods, only information on gene expression at the moment when the sample is prepared can be obtained. That is, by taking a part of the same sample or sample group over time and analyzing it, it is possible to follow changes over time in gene expression level. For this reason, it was impossible to measure changes over time in samples or cell groups in a strict sense.

[Analysis of gene expression by photoprotein]
When the gene expression product is a protein that can be optically observed from the outside, it is possible to observe the biological sample alive.
It is possible to measure the amount of firefly luciferase protein expressed while culturing the sample by expressing the firefly luciferase protein in the cell and measuring the luminescence intensity from the sample while culturing in the presence of D-luciferin. (Non-Patent Document 1).

It is also possible to measure the amount of expressed protein by expressing a fluorescent protein such as GFP in a cell and measuring fluorescence by applying excitation light (Non-patent Document 2).
Even when the expression product is RNA, a method has been devised for observing a biological sample alive with a combination of a specific RNA sequence and a chromophore that emits fluorescence when bound to the specific RNA sequence (Non-patent Document 3).

By inserting the DNA sequences encoding these detection proteins and RNA sequences into appropriate positions downstream of the promoter of the gene whose expression level is to be examined, the amount of the expression product can be observed by the luminescence phenomenon. .
By using these methods to record gene expression as a luminescence phenomenon in an image, it is possible to measure the spatial distribution of gene expression in an individual, tissue, or cell population while keeping a biological sample alive. . Furthermore, it is also possible to measure the temporal and spatial distribution of gene expression by taking images over time.

However, although it became possible to measure the temporal and spatial distribution of gene expression, a general-purpose method for quantitatively analyzing how the distribution changes has not been established. .
Except for special cases, researchers have analyzed their own methods according to the phenomenon, such as normalizing and displaying the temporal changes in signal intensity at multiple points on the image where the gene expression is recorded as a luminescent phenomenon. It is a situation.
As a special example, there is a clock gene whose expression level increases or decreases at a constant cycle over a long period of time. In the case of this clock gene, it is possible to define the phase of the increasing / decreasing period. Therefore, a method for analyzing the movement of the same phase region by a mathematical model and an example of analyzing a phase movement along a specific line segment in an image are known.

[Analysis of mass transfer speed]
The method of deriving the moving speed of a substance from an image has been known for a long time.
In the field of fluid dynamics, a method of mixing a tracking particle (tracer) in a fluid to be analyzed and analyzing a flow velocity distribution from the position of the tracking particle image in the image is well known as a particle image velocimetry (non-patent document). 4).
A method of tracking the movement of the same particle over time is called a particle tracking method.

[Image correlation method]
On the other hand, there is a method for obtaining the flow rate of a substance from the calculation of an image correlation function, which is called an image correlation method.
The image correlation method includes a method of calculating an autocorrelation function of images double-exposed on the same image sensor and a method of calculating a cross-correlation function of two images taken at different times. Today, with the advancement of image sensors, it has become easy to acquire images at high speed, and therefore it is mainstream to use a cross-correlation function.

In the field of meteorology, it is used as a method for measuring the wind speed at the position and altitude where clouds exist from analysis of cloud images of satellite photographs (Non-patent Document 5).
As a part of the image analysis method of the microscopic image, the image correlation method is applied to the biological image (Non-Patent Document 6).
The fluorescent molecules in the solution are constantly moving due to Brownian motion. As a technique for analyzing this motion, fluorescence correlation spectroscopy (FCS) has been developed.

The FCS collects excitation light in a sample solution using an optical system of a microscope, and measures the fluorescence intensity from the collected small area. The fluorescence intensity from the collected area is constantly fluctuating due to the movement of many fluorescent molecules in the collected area. By calculating and analyzing the autocorrelation function of the fluorescence intensity value with respect to time, the concentration and diffusion coefficient of the fluorescent molecule in the focal region can be estimated.
A series of techniques obtained by extending the FCS technique to an image of a scanning microscope or a total reflection microscope is called image correlation spectroscopy.

  Weisman et al. Developed Spatio-temporal Image Correlation Spectroscopy (STICS) in order to analyze the movement of scaffold proteins accompanying cell migration (Non-patent Document 7).

J. R. de Wet et al. (1987), “Firefly luciferase gene: structure and expression in mammalian cells”, Mol. Cell. Biol. 7, 725-737. M. Chalfie et al. (1994), “Green fluorescent protein as a marker for gene expression”, Science 263, 802-805. J. S. Paige et al. (2011), “RNA mimics of green fluorescent protein”, Science 333, 642-646. A. K. Prasad (2000), “Particle image velocimetry”, Current Sci. 79, 51-60. J. A. Leese et al. (1971), “An automated technique for obtaining cloud motion from geosynchronous satellite data using cross correlation”, J. Appl. Meteol. 10, 118-132. D. L. Kolin and P. W. Wiseman (2007), “Advances in image correlation spectroscopy: measuring number, aggregation states, and dynamics of fluorescently labeled macromolecules in cells”, Cell Biochem. Biophys. 49, 141-164. B. Hebert et al. (2005), “Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells”, Biophys. J. 88, 3601-3614. N. O. Petersen et al. (1993), “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application”, Biophys. J. 65, 1135-1146.

  The present invention has been made in view of the lack of a general-purpose method for quantitatively analyzing the temporal and spatial distribution of gene expression as described above, and is a biological sample taken over time. It is an object of the present invention to provide an analysis method that can be applied universally to a temporal change in gene expression distribution from a spatial distribution image of gene expression without limiting the phenomenon or target.

  It is another object of the present invention to provide an index for gene expression distribution of a sample and to provide a method for evaluating the influence of the environment in which the sample is placed on the gene expression of the sample.

According to one aspect of the present invention for solving the above-described problem , the biological sample image analysis method acquires distribution images of gene expression in cells of a plurality of biological samples photographed over time, and each acquired A plurality of small areas are set in the image, two arbitrary images are selected from the acquired plurality of images, and the correlation between the images of the corresponding two small areas is selected for the two selected images. A biological sample image analysis method for calculating a small region, repeatedly performing the image selection and the correlation calculation, and analyzing the movement speed and direction of gene expression for each small region , A first distribution image of gene expression in cells of a plurality of biological samples photographed over time, and a second distribution image related to cells of a plurality of biological samples photographed simultaneously with the first distribution images. Apply the above image analysis method , First and second velocity vectors each consisting of a moving speed and a moving direction for each small area are obtained from the first and second distribution images, and the first velocity vector and the second velocity vector are obtained. An operation related to the relationship is performed .

  According to the present invention, it is possible to provide a general-purpose technique for quantitatively analyzing the temporal and spatial distribution of gene expression.

The figure for demonstrating the image-analysis method of 1st Embodiment. The figure for demonstrating an example of the analysis method of 1st Embodiment. The figure for demonstrating the example which applied the image analysis method of 1st Embodiment to the observation of Arabidopsis thaliana. The figure which shows the image of the transmitted light which image | photographed continuously the process of embryonic development of the fruit fly egg. FIG. 5 is a diagram showing the result of image analysis processing of the transmitted light image shown in FIG. 4 superimposed on the original transmitted image. The figure which shows the image of the green light emission which image | photographed continuously the process of embryonic development of the fruit fly egg which was inject | poured the plasmid solution. FIG. 7 is a diagram showing the result of image analysis processing of the green light emission image shown in FIG. 6 superimposed on the original green light emission image. The figure which shows the image of the red light emission which image | photographed continuously the process of embryonic development of the fruit fly egg which was inject | poured the plasmid solution. The figure which shows the result of image-analysis processing of the red light emission image shown in FIG. 8 superimposed on the original red light emission image. The figure for demonstrating the correlation of the movement of the expression distribution of armadillo and shaggy using the image-analysis method of 1st Embodiment. The figure which shows the example which removes the influence of a cell movement from the movement of the expression distribution of each gene using the image analysis method of 1st Embodiment. The figure which shows the example which analyzed distribution of the moving speed of expression using the image analysis method of 1st Embodiment. 1 is a diagram illustrating a configuration of an image analysis system including an image analysis apparatus according to a first embodiment. The flowchart which shows the analysis process sequence of the image analyzer of 1st Embodiment.

[First Embodiment]
The expression of the genes described in the embodiments of the present invention is a phenomenon that occurs in individual cells. However, they influence neighboring cells by intercellular communication mechanisms such as the aforementioned signal transduction mechanisms. The expression distribution changes as it propagates. The present invention provides a technique for analyzing the change in the gene expression distribution of a biological sample using an image correlation method.
The present invention is not limited to the image correlation method described below. The image correlation method described below may be modified as appropriate, or another method including processing for obtaining correlation between images may be used.

  In the first embodiment, for the sake of explanation, the spatial coordinates x and y and the time coordinate t of a continuous image are handled as integers. By multiplying the proportionality factor determined from the photographing magnification and the photographing interval, it is possible to easily convert the actual position of the sample and the photographing time. Further, although the image is assumed to be two-dimensional for the sake of explanation, it can be easily extended to a three-dimensional image having stereoscopic information photographed by a confocal microscope or the like.

FIG. 1 is a diagram for explaining an image analysis method according to the first embodiment.
A biological sample is photographed at a fixed time interval with an image having a size of M × M (pixels), and N consecutive images are acquired. Image correlation calculation is performed on two (one set) images having a time interval τ from the N consecutive images. From N consecutive images, image correlation calculation is performed on (N−τ) sets of images. FIG. 1 shows a case where τ = 2.
For the sake of explanation, the correlation image has been translated so that the center of the image is (0, 0), but any coordinate system may be used depending on the calculation method.

Next, an average image correlation image obtained by averaging (N−τ) correlation images obtained by the image correlation calculation is obtained. The average image correlation image can be obtained by adding the image data of the corresponding pixel positions of each correlation image and calculating the average value.
The above process is expressed by a mathematical expression.
When the signal intensity of the image at the coordinates (x, y) acquired at time t is I (x, y; t), the average image correlation is expressed by Equation (1).

<I (x, y; t)> in the equations (1) and (2) indicates that the image data is averaged with respect to x and y of an image having a size of M × M (pixels). . That is, for the function F of x, y, t, <F (x, y; t)> is defined by equation (3).

  For x and y, if F (x + nM, y + mM; t) = F (x, y; t) is satisfied for the integers n and m, the numerator of formula (1) The described cross-correlation function can be calculated at high speed using Fourier transform.

In addition, in order to catch the movement of the expression distribution in the sample with time, it is possible to remove in advance a component that is immobile or a component that is slow in the time segment to be analyzed. As a removal method, as described in (Non-Patent Document 7), the low frequency component of the signal intensity change may be removed for each coordinate. Or you may remove a stationary component so that it may be guessed immediately from (nonpatent literature 8). That is, the moving component I _mov may be calculated according to Equation (4), and I _mov may be used instead of I (x, y; t) in Equation (1).

Next, an analysis method using the average image correlation image will be described.
FIG. 2 is a diagram for explaining an example of the analysis method according to the first embodiment.
When the distribution image in the continuous image moves in a specific direction with time, the average image correlation image R (ξ, η, τ) obtained by the above method has a maximum value at a position deviated from the center of the image. It becomes a distribution image. As one method for determining the position of the local maximum value, there is a method of fitting a cross-correlation image assuming a Gaussian distribution.

That is, the Gaussian represented by the equation (5) is fitted to the average image correlation image by the least square method with A, β, p, q, and C as variable parameters.

統計的に正確なｖ_ｘ、ｖ_ｙを導出するため、図２に示すように、最小二乗法などを用いた回帰分析によって比例係数ｖ_ｘ、ｖ_ｙを算出しても良い。 P and q obtained by fitting are the maximum positions (p _τ and q _τ ) of the average image correlation image in the time interval τ. Therefore, the maximum position (p _τ , q _τ ) of the average image correlation image is determined for each delay time τ (= 1 to N−1).
[Moving Speed]
The moving speed of the distribution in the image _(v x, _{v y),} the proportional coefficient of the delay time and maximum position of the formula (6) _v x, is determined as _{v y.}

In order to derive statistically accurate v _x and v _y , as shown in FIG. 2, the proportional coefficients v _x and v _y may be calculated by regression analysis using a least square method or the like.

[Movement speed distribution]
By performing the above analysis on the image in the m × m (pixel) partial area included in the entire M × M (pixel) image, the local movement speed in the partial area is derived. I can do it. That is, image correlation is calculated for the partial area A _i where x is from x _i to x _i + m−1 and y is from y _i to y _i + m−1, and the moving speed for the partial area is determined. it can. By obtaining the local moving speed for a large number of partial images, the moving speed distribution can be obtained.

[Analysis over time]
The moving speed at the time point t can be derived by taking out a time segment consisting of n images closest to the time t from a plurality of images having different shooting times and performing the above analysis. That is, the image correlation is calculated for an image in which t is from t _i to t _i + n−1, and is determined as, for example, the moving speed at the time t = t _i + (n−1) / 2 at the center of this time segment. can do. By obtaining the moving speed by shifting the time segments continuously or discretely, the moving speed changing can be grasped, and the moving speed can be analyzed over time.

[Vector display]
The moving speed obtained by the above analysis method is displayed on the screen, thereby visualizing the result and assisting the user in visually understanding the result. As a display method, for example, as shown in the lower right of FIG. 2, a method of superimposing and displaying a graphic representing a velocity vector on a continuous image is conceivable. As a figure representing a velocity vector, there is an arrow whose length is proportional to the magnitude of the moving velocity and whose arrowhead indicates the direction of the vector. Also, the color of the arrow can be displayed according to the magnitude of the speed.

  When analysis is performed in a partial region in the image and the movement speed of the local expression distribution is analyzed, the velocity distribution can be visualized by displaying a vector in the center of each partial region. When analysis over time is performed, the temporal change of the moving speed can be visualized by displaying the speed vector at the time t superimposed on the image at the time t.

[Vector operation]
The calculation is performed between the movement speed vector v ₁ of the gene expression distribution obtained by the image correlation method and another vector v ₂ at the time / coordinate obtained for another analysis object, and the operation result is obtained at the time / coordinate. The analysis result can be used.

The vector v ₂ for another analyzed, the moving velocity vector different genes Gene expression profile derived from the expression distribution (gene 2) is considered a gene (gene 1), for example v ₁ to be analyzed. As a method of obtaining expression distribution images of different genes from the same sample at the same time, a method of expressing photoproteins that emit light of different wavelengths from the respective genes can be considered.

Other examples obtaining a vector v ₂ for another analyzed, the moving velocity vector of the cells derived from the image of the cell distribution are contemplated. As a method of obtaining the gene expression distribution image and the cell distribution image from the same sample, a method of switching the optical system of the photographing apparatus and photographing the cell distribution by a differential interference microscope is conceivable.

As an example of an operation between vectors, an inner product operation of two vectors can be considered. The inner product of the movement speed vectors v1 and v2 of the expression distributions of the two genes is expressed by Expression (7), and the cosine of the angle formed by the movement speed vectors v1 and v2 is expressed by Expression (8).

  As described above, a plurality of genes form a network that controls the expression of each other. Therefore, the expression distribution of genes downstream of the network is a distribution dependent on the expression distribution of upstream genes. That is, whether or not there is a correlation in the direction of movement of the gene expression distribution can be an indicator of whether or not there is a relationship between the two gene expressions. Therefore, the correlation in the moving direction can be expressed by using the cosine of the angle θ formed by the two vectors as an index.

As another example of calculation between vectors, subtraction between vectors can be considered. An example in which subtraction between vectors is effective is calculation between a moving speed vector of a gene expression distribution and a moving speed vector of a cell containing the gene.
When cells actively divide and move like embryogenesis, even if the gene expression level in the cell is constant, the expression distribution moves with the movement of the cell. Therefore, in order to eliminate the influence of cell migration from the movement of the gene expression profile, it subtracts the moving velocity vector v ₂ of the cell from moving velocity vector v ₁ of the gene expression distribution. By this calculation, the absolute movement velocity vector v _rel (= v ₁ −v ₂ ) of gene expression can be calculated.

[Indicators of gene expression]
The information obtained by analyzing and quantifying the temporal change in the gene expression distribution of the biological sample described above can be used as an index of gene expression in the environment where the sample is placed.
For example, assume that there is a biological sample in which the promoter activity of a gene A can be detected by the expression of a photoprotein. By applying the above-described method to the luminescence microscopic image of this biological sample, it is possible to analyze the temporal change in the gene expression distribution of gene A. By adding various reagents for the purpose of promoting or suppressing the expression of gene A to the culture solution of this biological sample, and analyzing and comparing the changes in the gene expression distribution over time, it can be understood by simply increasing or decreasing the expression level. Higher level information that did not exist can be obtained.

As an index of a change in gene expression distribution, for example, there is a statistic obtained by statistically processing a velocity vector distribution. The image is divided into small partial areas, and the velocity vector distribution is obtained by the analysis method of the present invention. Then, the influence of the environment can be evaluated using the average value or median value of the velocity magnitude distribution {| v _i |} as one of the gene expression indexes in the environment where the biological sample is placed.

Subsequently, an embodiment to which the above analysis method is applied will be described.
[Example 1 (plant tissue / biological rhythm)]
FIG. 3 is a diagram for explaining an example in which the image analysis method according to the first embodiment is applied to observation of Arabidopsis thaliana.
In this example, a gene expression network via a clock gene is observed for a plant (Arabidopsis thaliana). Leaves were excised from the Arabidopsis pcab2: luc strain grown for 2 weeks in a light-dark cycle (13 hours light / 11 hours dark) and placed on an agarose gel and sprayed with 2.5 mM D-luciferin. Changes in the luminescence signal were continuously observed every 2 hours with an upright luminescence microscope.

  As shown in the continuous images at the top of FIG. 3, firefly luciferase expressed in Arabidopsis thaliana in the presence of luciferin emits light. This firefly luciferase is produced with the expression of the cab2 gene. The change in the luminescence state of luciferase with time represents the transition of the expression level of the cab2 gene.

About the continuous image of the light emission image obtained in this way, a 32 × 32 small region (ROI: Region of Interest) is vertically and horizontally oriented with respect to the continuous image composed of 16 square images each having 340 pixels. Each ROI was processed while shifting 6 pixels at a time.
Processing in each ROI was performed as follows.

Step 1: Calculate cross-correlation images for two images with a time interval τ from τ = 1 to 5 (sheets), 15 for τ = 1, 14 for τ = 2, and so on. A cross-correlation average image was obtained by averaging (16−τ) sheets.
Step 2: The position of the peak value was obtained from the cross-correlation average image for each τ value. The position of the peak value was determined from the position of the Gaussian vertex by fitting a two-dimensional Gaussian function to the original image by the method of least squares.

Step 3: From the position of the peak (x _τ , y _τ ) for each τ value, the signal transfer speed at that ROI was derived. The x-axis component value v _x of the signal movement speed was derived by applying x _τ = v _x × τ to the x-axis peak position x _τ for each τ value by the least square method. For the y-axis component, _vy was derived by the same method.

Step 4: When the peak position could not be identified, the speed was derived based on the data in the range of the identified time interval. If the peak position could not be identified for τ> 0, it was considered that there was no significant velocity component, and the velocity component for that ROI was undefined.
Step 5: The signal moving speed in each ROI was displayed by drawing an arrow having a length proportional to the magnitude of the speed in the direction of the speed from the center coordinates of the ROI.

  The lower part of FIG. 3 shows a diagram in which an arrow indicating the signal moving speed is overlaid on a continuous image. The size of the arrow can be arbitrarily selected so that the display is easy to see, but in the case of this image, the speed component of 1 pixel / sheet was selected to be displayed with a length of 4 pixels. The color of the arrow indicating the speed was colored from purple to red according to the color of the rainbow from the smallest speed component to the largest. Then, the correspondence between the speed value on the actual scale calculated from the magnification of the image and the acquisition time interval and the length / color of the arrow is expressed by displaying the corresponding scale at an arbitrary position on the image.

The situation in which a specific gene is generated can be grasped in more detail by synthesizing and drawing an arrow representing the signal movement speed on a continuous image.
In the figure, the portion that glows white near the center contains the veins of Arabidopsis thaliana. Then, it can be understood that the signal is moving so as to spread outward from the veins. Further, the transmission time can be estimated from the speed and distance represented by the arrows.

Therefore, by applying this analysis method, it is possible to grasp in more detail how the signal is transferred, not just determining the presence or absence of gene expression in a certain time section.
[Example 2 (Animal tissue / embryo development)]
Next, an example in which the image analysis method according to the first embodiment is applied to Drosophila observation will be described with reference to FIGS.

  An aqueous solution containing two expression plasmids (two expression plasmids each having a concentration of 200 ng / μL, 2.5 mM D-luciferin, 0.5% brilliant blue) at the tail of the egg immediately after egg laying in Drosophila FCF) was injected with a microinjector. One of the expression plasmids is one in which a 1.8 kb armadillo promoter cloned from Drosophila is inserted upstream of the CBR gene of the commercially available expression plasmid pCBR-Basic (Promega). One is a 3.0 kb shaggy promoter cloned from Drosophila inserted upstream of the CBG gene of the commercially available expression plasmid pCBG99-Basic (Promega).

  The Drosophila eggs after the injection of the plasmid solution were allowed to stand in an observation container kept at 25 ° C. and humidified, and the embryogenesis process was continuously photographed with a luminescence microscope LUMINOWEW LV200 (manufactured by Olympus). The objective lens was 60 times the NA value of 1.25, and the camera was an ImagEM EM-CCD camera (Hamamatsu Photonics). Photographing was performed at intervals of 10 minutes while automatically switching between the optical system and the filter for illumination transmitted light and green and red light emission. The transmitted light was photographed by 170 millisecond exposure, the green light by BP515-560 bandpass filter using 390 seconds exposure, and the red light using 610ALP longpass filter.

With regard to the continuous image composed of 150 images of 512 × 256 pixels of each of the three types of emission images obtained in this way, the processing is performed for each ROI while shifting the 64 × 64 ROI by 8 pixels for the emission channel. Executed.
The processing in each ROI is carried out by performing image analysis on 15 images of 64 × 64 pixels cut out from the same position of 15 consecutive images from 150 images, and obtaining 15 vector values obtained from the analysis. The moving speed at the time of the eighth sheet, which is the center of.

FIG. 4 is a view showing transmitted light images obtained by continuously photographing the Drosophila egg embryo development process. In these images, cells in the early stage of embryogenesis (represented by white dots in the figure) divide and move in the egg.
FIG. 5 is a diagram showing the result of image analysis processing of the transmitted light image shown in FIG. 4 superimposed on the original transmitted image. For example, when an attempt is made to express a phenomenon such as the appearance of cells that form the intestine by referring to only the transmission image, the phenomenon is expressed descriptively. In contrast, by synthesizing and expressing the transmitted light image and the result of image processing, the movement of the cell can be grasped in detail and quantitatively, so that the phenomenon can be expressed more accurately.

FIG. 6 is a diagram showing green luminescence images obtained by continuously photographing embryonic development of Drosophila eggs injected with a plasmid solution.
As described above, a synthetic gene in which a CBG (green luciferase-derived green luminescence luciferase) gene is connected downstream of the shaggy promoter is prepared and injected into a Drosophila egg. In Drosophila eggs, shaggy is generated (replicated) during embryogenesis, and the injected synthetic gene is also replicated. In the continuous image shown in FIG. 6, the situation where the injected synthetic gene is replicated is represented as a green region.

FIG. 7 is a diagram showing the result of image analysis processing of the green light emission image shown in FIG. 6 superimposed on the original green light emission image. As a result, the situation where the shaggy is generated can be grasped together with its movement (direction, speed, etc.).
FIG. 8 is a diagram showing red luminescence images obtained by continuously photographing the embryogenesis process of Drosophila eggs injected with a plasmid solution.
As described above, a synthetic gene in which a CBR (red light luciferase-derived red luciferase) gene is connected downstream of the armadillo promoter is generated and injected into a Drosophila egg. In Drosophila eggs, it is said that when staggy is produced during embryogenesis, armadillo is produced. In the continuous image shown in FIG. 8, the situation in which armadillo is generated is represented as a red region.

FIG. 9 is a diagram showing the result of image analysis processing of the red light emission image shown in FIG. 8 superimposed on the original red light emission image. As a result, the situation in which armadillo is generated can be grasped together with its movement (direction, speed, etc.).
[Correlation of velocity distribution]
FIG. 10 is a diagram for explaining the correlation of the movement of the expression distribution of armadillo and shaggy using the image analysis method of the first embodiment.

  (1) of FIG. 10 shows a result of obtaining a velocity vector by analyzing a continuous image in the vicinity of 7:30 of armadillo shown in FIG. (2) of FIG. 10 shows a result of obtaining a velocity vector by analyzing a continuous image around 7:30 of shaggy shown in FIG.

  (3) in FIG. 10 represents the correlation between the directions of the velocity vectors for each ROI using the cosine of the angle formed by the two velocity vectors (equation (8)). FIG. 10 (3) shows that the cosine of the angle formed by most two velocity vectors = + 1, that is, the two velocity vectors have the same size and the same moving direction. Therefore, it is confirmed that when shaggy is generated, armadillo is generated concomitantly.

[Relative movement speed]
FIG. 11 is a diagram illustrating an example in which the influence of cell movement is removed from the movement of the expression distribution of each gene using the image analysis method according to the first embodiment.
(1) in FIG. 11 shows the result of calculating the velocity vector by analyzing nine consecutive images in the vicinity of 7:30 of the shaggy shown in FIG. (2) in FIG. 11 shows the result of calculating the velocity vector by analyzing nine consecutive images near 7:30 of the transmitted light image shown in FIG.

(3) in FIG. 11 shows the result of subtracting the velocity vector of the transmitted light image from the shaggy velocity vector for each ROI. Thereby, the movement speed of substantial gene expression distribution can be evaluated.
[Index of expression]
FIG. 12 is a diagram illustrating an example in which the distribution of the movement speed of expression is analyzed using the image analysis method according to the first embodiment.
As a result of analyzing the magnitude distribution of the moving speed of the speed vector in the vicinity of 7:30 of shaggy shown in FIG. 7, the median value of the distribution = 18 μm / h was obtained.

  Conventionally, as an index of gene expression, the expression level of a whole sample or the expression level per cell after separately measuring the number of cells has been required. However, there are cases where it is not sufficient as an index for judging minute differences due to changes in gene culture medium or reagents.

On the other hand, by using this analysis result, it is possible to obtain highly accurate supporting data when performing such an evaluation. For example, it is possible to make a quantitative evaluation using statistics such as a change in maximum speed, a change in median value, a change in distribution shape, and a change in deviation value.
The image analysis method described above can be performed not only manually (manually) but also automatically by an image analysis apparatus equipped with the image analysis processing function. In the following, modes that are automatically implemented will be described.

FIG. 13 is a diagram illustrating a configuration of an image analysis system including the image analysis apparatus according to the first embodiment.
The image analysis system 1 includes an image capturing device 2, an image analysis device 3, and an input / output device 4. The image capturing device 2 captures a biological sample in accordance with an operation instruction from the image analyzing device 3 and outputs the captured image to the image analyzing device 3. The image analysis device 3 analyzes the image captured by the image capturing device 2 and analyzes temporal changes in the gene expression distribution of the biological sample. The input / output device 4 displays the result analyzed by the image analysis device 3 and gives various operation instructions to the image analysis device 3 based on the operation input of the operator.

The image analysis device 3 includes an input / output interface 11, a shooting control unit 12, a shot image acquisition unit 13, an image analysis unit 14, an editing output unit 15, and a storage unit 16.
The input / output interface 11 is an interface for exchanging information between the image capturing device 2 and the input / output device 4. The shooting control unit 12 instructs the image shooting apparatus 2 to perform a shooting operation. The captured image acquisition unit 13 acquires a continuous image obtained by capturing a biological sample from the image capturing device 2 and stores it in the storage unit 16. The image analysis unit 14 calculates various data representing temporal changes in gene expression distribution of biological samples by analyzing continuous images. The edit output unit 15 edits the analysis result and outputs it to the input / output device 4. The storage unit 16 stores data such as continuous images and analysis result edited images.

Next, the operation of the image analysis system will be described.
The operator inputs operating conditions of the image capturing device 2 from the input / output device 4. For example, information such as shooting time, shooting period, shooting interval, number of shots, shooting conditions (light source type, exposure time, filter type, etc.), type of optical system to be used, and the like are input. The imaging control unit 12 controls the image capturing device 2 according to the input operation condition to capture a biological sample, and outputs a continuous image to the image analysis device 3. The captured image acquisition unit 13 receives a continuous image sent from the image capturing device 2 and stores it in the storage unit 16 together with information for identifying the continuous image.

When a predetermined shooting operation (for example, when shooting of one cycle of continuous images is completed) is executed, the image analysis unit 14 performs analysis of the continuous images stored in the storage unit 16.
FIG. 14 is a flowchart illustrating an analysis processing procedure of the image analysis apparatus according to the first embodiment.

  In step S <b> 01, the image analysis unit 14 reads N consecutive images captured by the image capturing device 2 from the storage unit 16. In step S02, the image analysis unit 14 sets a value for the interval τ. Here, τ can take a natural number of 1 to (N−1). Then, the image analysis unit 14 takes out two consecutive images with an interval τ as an analysis target.

  In step S03, the image analysis unit 14 sets an ROI that is a small region at the same position in the two extracted continuous images. In step S04, the image analysis unit 14 obtains cross-correlation images of two ROIs. The method for obtaining the cross-correlation image is not limited to Equation (1), and various known methods may be used. In step S05, the image analysis unit 14 acquires the coordinates of the peak position from the cross-correlation image. As a technique for acquiring the coordinates of the peak position, there is a fitting method in which fitting is performed on the cross-correlation image assuming a Gaussian distribution.

  In step S06, it is checked whether or not the peak position coordinates have been acquired for all the ROIs. When the coordinates of the peak positions are not acquired for all ROIs (S06 NO), a new ROI is set by moving the ROI by a predetermined pixel in the vertical or horizontal direction of the continuous image, and this new ROI is targeted. Then, the processes of steps S04 and S05 are executed.

  When the coordinates of the peak positions are acquired for all the ROIs (S06 Yes), a new value is set for the interval τ in step S07, and the processing from step S03 to step S06 is executed. When the processing from step S03 to step S06 is executed for all possible values of the interval τ (Yes in S07), in step S08, velocity vectors (moving speed, moving direction) are obtained for all ROIs. In step S09, the image analysis unit 09 stores the velocity vector for each ROI in the storage unit 16 in association with the continuous image. Note that the operator sets the ROI size used in the above analysis, the number of pixels to which the ROI is moved, and the like from the input / output device 4 in advance.

When the operator inputs a request for outputting the analysis result from the input / output device 4 after the imaging of the biological sample is completed, the editing output unit 15 edits the analysis result in accordance with the requested content and sends it to the input / output device 4. Output.
For example, the edit output unit 15 edits the following analysis result image and outputs it to the input / output device 4.

[Velocity distribution image]
A speed distribution image is edited in which an arrow representing a moving speed for each of a plurality of partial areas included in the whole image is superimposed on the whole image (continuous image) of the biological sample. Here, the length of the figure representing the arrow is proportional to the magnitude of the moving speed, and the arrowhead indicates the moving direction. The color of the arrow is displayed corresponding to the speed.

[Images over time]
The speed distribution image is created for each time segment. Then, the created velocity distribution image for each time segment is displayed as a moving image having one frame image.
[Relevance analysis image]
Whole images (continuous images) are taken with different imaging means for the same biological sample. Then, the above-described velocity distribution image is edited for each entire image. Then, vector analysis (cosine calculation, subtraction, etc. of the angle formed by the two velocity vectors) is performed on the movement speeds of the corresponding partial areas, and a relevance analysis image obtained by superimposing the analysis results on the entire image is displayed.

[Indicator value image]
The moving speed for each of the plurality of partial areas included in the entire image (continuous image) of the biological sample is obtained. Then, an index value is calculated by analysis (statistical analysis) from the obtained plurality of moving speeds. Then, an index value image representing the calculated index value is displayed.
According to the image analysis system described above, an analysis result can be obtained in a short time (immediately) after the imaging of the biological sample is completed.
In the above-described embodiment, the image capturing device 2 and the image analysis device 3 are configured separately. However, the image capturing device 2 may be equipped with these functions as a unit.

[Variation of the first embodiment]
Note that the content disclosed in the first embodiment described above is not limited to the disclosed mode, and can be configured as various variations.
In the above-described embodiment, the continuous image is two-dimensional, but may be a three-dimensional image having stereoscopic information photographed by a confocal microscope or the like. Therefore, the imaging apparatus can be grasped as an apparatus that records an image representing a two-dimensional space or a three-dimensional space, that is, a distribution of gene expression in spatial coordinates of a biological sample.

  In addition, gene expression in cells is not only observed by fluorescence but also by light emission using luciferase or the like, and thus can generally be grasped as a luminescence phenomenon. Therefore, gene expression in the cell is acquired as a difference in luminescence intensity from the cell.

In addition, the protein that emits light in the above-described embodiment is not limited to proteins generally called fluorescent protein and photoprotein unless otherwise specified.
In addition, the protein that emits light in the above-described embodiment may be provided in a site serving as a marker for expressing a gene (promoter analysis).

  In addition to the aspect in which the entire protein is provided at the site that serves as a marker for gene expression, the protein that emits light is a protein that emits light in addition to the protein that the biological sample originally has in the downstream of the promoter. It can exist in an embodiment (chimeric protein or fusion protein).

[Effect of the embodiment]
According to the above-described embodiment, the temporal change in gene expression distribution can be applied universally without limiting the phenomenon or target from the spatial distribution image of gene expression in biological samples taken over time. Can provide a way. In addition, it is possible to provide an index for the gene expression distribution of the sample and provide a method for evaluating the influence of the environment in which the sample is placed on the gene expression of the sample.
Therefore, according to the present embodiment, it is possible to provide a general-purpose technique for quantitatively analyzing the temporal and spatial distribution of gene expression.

In addition, according to the present invention, it is possible to perform general-purpose and quantitative analysis when analyzing gene expression distribution of a biological sample in biological research or medical examination / diagnosis. As a result, it becomes possible to handle the phenomenon quantitatively and statistically, so that it is possible to discover a phenomenon that has been missed by the conventional method. Further, according to the present invention, it is possible to visualize the phenomenon of gene expression distribution movement. As a result, the person performing the experiment helps to understand the phenomenon indicated by the experiment result, and facilitates information exchange with others.
Note that the functions described in the above-described embodiments are not limited to being configured using hardware, and can be realized by causing a computer to read a program describing each function using software. Each function may be configured by appropriately selecting either software or hardware.

Note that the present invention is not limited to the above-described embodiment as it is, and can be embodied by modifying the constituent elements without departing from the scope of the invention in the implementation stage.
Various inventions can be formed by appropriately combining a plurality of constituent elements disclosed in the embodiment. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, you may combine suitably the component covering different embodiment.

  The present invention can be used in industries that provide a general-purpose technique for quantitatively analyzing the temporal and spatial distribution of gene expression.

  DESCRIPTION OF SYMBOLS 1 ... Image analysis system, 2 ... Image imaging device, 3 ... Image analysis device, 4 ... Input / output device, 11 ... Input / output interface, 12 ... Shooting control part, 13 ... Captured image acquisition part, 14 ... Image analysis part, 15 ... edit output unit, 16 ... storage unit.

Claims (12)

Obtain distribution images of gene expression in cells of multiple biological samples taken over time,
Set multiple small areas in each acquired image,
Select any two images from the acquired images,
For the two selected images, the correlation between the images of the corresponding two small areas is calculated for a plurality of small areas,
Analyzing the movement speed and direction of gene expression for each small region by repeatedly executing the image selection and correlation calculation
An image analysis method for biological samples,
A first distribution image of gene expression in cells of a plurality of biological samples photographed over time, and a second distribution image related to cells of a plurality of biological samples photographed simultaneously with the first distribution images. Applying the above image analysis method,
First and second velocity vectors each consisting of a moving speed and a moving direction for each small region are obtained from the first and second distribution images,
A biological sample image analysis method, wherein an operation relating to a relationship between the first velocity vector and the second velocity vector is performed . The second distribution image is an image showing the cells of the biological sample;
The calculation related to the relationship between the first speed vector and the second speed vector is performed by subtracting the second speed vector from the first speed vector to obtain a new moving speed and moving direction for each small area. Is an operation for obtaining
The biological sample image analysis method according to claim 1. The second distribution image is an image showing gene expression in a cell of the biological sample, which is different from the gene expression according to the first distribution image,
The calculation related to the relationship between the first speed vector and the second speed vector is a calculation of the correlation between the first speed vector and the second speed vector.
The biological sample image analysis method according to claim 1.   2. The biological sample according to claim 1, wherein a composite image in which an arrow image representing a movement speed and a movement direction of gene expression for each small region is superimposed on a corresponding position on the distribution image is generated. Image analysis method.   The value obtained by statistically processing the migration speed of gene expression for each small region obtained by the analysis is output as an index of the gene expression in an environment where the biological sample is placed. Biological sample image analysis method.   The biological sample image analysis method according to any one of claims 1 to 5, wherein gene expression in a cell is expressed as luminescence intensity in the photographed image.   The method for analyzing an image of a biological sample according to claim 6, wherein the luminescence from the cell is luminescence from a protein expressed in the cell. The biological protein image analysis method according to claim 7, wherein the expressed protein is under the control of a promoter of a gene to be analyzed.   9. The method for analyzing an image of a biological sample according to claim 8, wherein the protein under the promoter control of the gene contains all or part of the photoprotein. An acquisition means for acquiring a distribution image of gene expression in cells of a plurality of biological samples photographed over time,
A setting means for setting a plurality of small regions in each acquired image;
Selection means for selecting any two images from the plurality of acquired images;
Calculating means for calculating the correlation between the images of the two corresponding small areas for the selected two images for a plurality of small areas;
Analyzing means for repeatedly executing the image selection and correlation calculation, and analyzing the movement speed and movement direction of gene expression for each small region ,
The acquisition means includes a first distribution image of gene expression in cells of a plurality of biological samples photographed over time, and a second relating to cells of the plurality of biological samples photographed simultaneously with the first distribution images. And get a distribution image of
The setting means, the selection means, the calculation means, and the analysis means respectively obtain first and second velocity vectors composed of a movement speed and a movement direction for each small region from the first and second distribution images. An image analysis apparatus for a biological sample, characterized in that the calculation is performed and a calculation related to the relationship between the first velocity vector and the second velocity vector is performed . Computer
11. A computer program that functions as an acquisition unit, a setting unit, a selection unit, a calculation unit, and an analysis unit provided in the image analysis apparatus according to claim 10. Photographing means for photographing distribution images of gene expression in cells of biological samples over time;
Acquisition means for acquiring a plurality of photographed distribution images;
A setting means for setting a plurality of small regions in each acquired image;
Selection means for selecting any two images from the plurality of acquired images;
Calculating means for calculating the correlation between the images of the two corresponding small areas for the selected two images for a plurality of small areas;
Analyzing means for repeatedly executing the image selection and correlation calculation, and analyzing the movement speed and movement direction of gene expression for each small region ,
The imaging means includes a first distribution image of gene expression in cells of a plurality of biological samples photographed over time, and a second relating to cells of the plurality of biological samples photographed simultaneously with the first distribution images. Take a distribution image of
The acquisition means acquires the captured first distribution image and the second distribution image,
The setting means, the selection means, the calculation means, and the analysis means respectively obtain first and second velocity vectors composed of a movement speed and a movement direction for each small region from the first and second distribution images. An image photographing apparatus for a biological sample, characterized in that a calculation is performed on the relationship between the first velocity vector and the second velocity vector .

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