JP2008212017A5

JP2008212017A5 -

Info

Publication number: JP2008212017A5
Application number: JP2007051150A
Authority: JP
Filing date: 2007-03-01
Publication date: 2011-03-31

Description

本発明は、細胞培養容器内で培養されている浮遊細胞を観察するための位相差顕微鏡を用いて観察した前記細胞培養容器内の画像（位相差画像）を撮像して取得し、取得した位相差画像に対して二次微分フィルタを適用してエッジを抽出し、抽出したエッジが構成する図形の面積が最大のもの、またはエッジが構成する図形の輪郭が最長のものを浮遊細胞の形状として抽出し、浮遊細胞の形状に基づいて、浮遊細胞の活性状態を判定することを特徴とする。
本発明では、浮遊細胞の形状の円形度合いに基づいて、浮遊細胞の活性状態を判定することが好ましい。このとき、（１）エッジの持つ共分散行列の固有値を用いて算出される浮遊細胞の形状の楕円度、（２）浮遊細胞の形状の重心からエッジまでの距離の最大値−最小値、または当該距離の分散により算出される浮遊細胞の形状の真円度、（３）浮遊細胞の形状の周囲長の２乗／面積により算出される浮遊細胞の形状の複雑度、および（４）４π×浮遊細胞の形状の面積／浮遊細胞の形状の周囲長の２乗により算出される浮遊細胞の形状の円形度の少なくとも１つを浮遊細胞の形状の円形度合いを表す指標として用い、これらの指標に基づいて浮遊細胞の活性状態を判定するようにしてもよい。
また、活性状態の判定結果を所定時間間隔で取得し、浮遊細胞の活性状態の時系列変化を観察するようにしてもよい。
本発明はまた、細胞培養容器内で培養されている浮遊細胞を観察するための位相差顕微鏡を用いて観察した細胞培養容器内の画像（位相差画像）を撮像して取得し、取得した位相差画像からエッジを抽出し、抽出したエッジが構成する図形の面積が最大のもの、またはエッジが構成する図形の輪郭が最長のものを浮遊細胞の形状として抽出し、浮遊細胞の形状に基づいて、浮遊細胞の活性状態を判定することを特徴とする。
The present invention captures and acquires an image (phase contrast image) in the cell culture container observed using a phase contrast microscope for observing floating cells cultured in the cell culture container. Edges are extracted by applying a second-order differential filter to the phase difference image, and the shape of the figure composed of the extracted edges is the largest, or the shape of the figure composed of the edges is the longest as the shape of the floating cell Extracting and determining the active state of the floating cells based on the shape of the floating cells.
In the present invention, it is preferable to determine the active state of the floating cell based on the circularity of the shape of the floating cell. At this time, (1) the ellipticity of the shape of the floating cell calculated using the eigenvalue of the covariance matrix of the edge, (2) the maximum value-minimum value of the distance from the center of gravity of the floating cell to the edge, or The roundness of the shape of the floating cell calculated by the dispersion of the distance, (3) the complexity of the shape of the floating cell calculated by the square of the perimeter of the shape of the floating cell / area, and (4) 4π × At least one of the circularity of the floating cell shape calculated by the square of the area of the floating cell shape / peripheral length of the floating cell shape is used as an index representing the circularity of the floating cell shape. Based on this, the active state of the floating cell may be determined.
Alternatively, the determination result of the active state may be acquired at predetermined time intervals, and the time series change of the active state of the floating cell may be observed.
The present invention also captures and acquires an image (phase contrast image) in a cell culture container observed using a phase contrast microscope for observing floating cells cultured in the cell culture container. Extract the edges from the phase difference image, extract the one with the largest area of the figure composed of the extracted edges or the one with the longest outline of the figure composed of the edges as the floating cell shape, and based on the shape of the floating cell The active state of floating cells is determined.

Claims

A phase contrast microscope for observing floating cells cultured in a cell culture vessel;
Imaging means for capturing and acquiring an image (phase difference image) in the cell culture vessel observed using the phase contrast microscope;
Edge extraction means for extracting an edge by applying a second-order differential filter to the phase difference image acquired by the imaging means;
Shape extraction means for extracting the one with the largest area of the figure formed by the edge extracted by the edge extraction means, or the one with the longest outline of the figure constituted by the edge as the shape of the floating cell;
A cell state determination device comprising: a determination unit that determines an active state of the floating cell based on the shape of the floating cell extracted by the shape extraction unit.

In the cell state determination apparatus according to claim 1,
The determination unit includes a determination unit that determines an active state of the floating cell based on a circular degree of the shape of the floating cell.

The cell state determination apparatus according to claim 2,
The determination means is (1) the ellipticity of the shape of the floating cell calculated using the eigenvalue of the covariance matrix of the edge, and (2) the maximum value of the distance from the center of gravity to the edge of the shape of the floating cell. The roundness of the shape of the floating cell calculated by the minimum value or the dispersion of the distance; (3) the complexity of the shape of the floating cell calculated by the square of the perimeter of the shape of the floating cell / area And (4) 4π × the area of the shape of the floating cell / the roundness of the shape of the floating cell calculated by the square of the circumference of the shape of the floating cell is at least one of the circularity of the shape of the floating cell. A cell state determination apparatus characterized in that it is used as an index representing the degree of circularity, and the active state of the floating cell is determined based on these indexes.

In the cell state determination apparatus according to any one of claims 1 to 3,
A cell state determination apparatus, further comprising an observation unit that obtains determination results by the determination unit at predetermined time intervals and observes a time-series change in the active state of the floating cells.

A phase contrast microscope for observing floating cells cultured in a cell culture vessel;
Imaging means for capturing and acquiring an image (phase difference image) in the cell culture vessel observed using the phase contrast microscope;
Edge extraction means for extracting an edge from the phase difference image acquired by the imaging means;
Shape extraction means for extracting the one with the largest area of the figure formed by the edge extracted by the edge extraction means, or the one with the longest outline of the figure constituted by the edge as the shape of the floating cell;
A cell state determination apparatus comprising: determination means for determining an active state of the floating cell based on the shape of the floating cell extracted by the shape extraction unit.

Obtain and acquire an image (phase contrast image) in the cell culture container observed using a phase contrast microscope for observing floating cells cultured in the cell culture container,
Applying a second-order differential filter to the acquired phase difference image to extract edges,
Extract the one that has the largest area of the figure formed by the extracted edge, or the one having the longest outline of the figure that the edge constitutes as the shape of the floating cell,
A cell state determination method, wherein an active state of the floating cell is determined based on a shape of the floating cell.

In the cell state determination method according to claim 6,
A cell state determination method, comprising: determining an active state of the floating cell based on a circularity of the shape of the floating cell.

The cell state determination method according to claim 7,
(1) The ellipticity of the shape of the floating cell calculated using the eigenvalue of the covariance matrix of the edge, (2) the maximum value-minimum value of the distance from the center of gravity of the floating cell shape to the edge, or The roundness of the shape of the floating cell calculated by the dispersion of the distance, (3) the complexity of the shape of the floating cell calculated by the square of the perimeter of the shape of the floating cell / area, and (4 ) 4π × the area of the shape of the floating cell / at least one of the circularity of the shape of the floating cell calculated by the square of the perimeter of the shape of the floating cell is an index representing the degree of circularity of the shape of the floating cell And determining the active state of the floating cell based on these indicators.

Obtain and acquire an image (phase contrast image) in the cell culture container observed using a phase contrast microscope for observing floating cells cultured in the cell culture container,
Edges are extracted from the acquired phase difference image,
Extract the one that has the largest area of the figure formed by the extracted edge, or the one having the longest outline of the figure that the edge constitutes as the shape of the floating cell,
A cell state determination method, wherein an active state of the floating cell is determined based on a shape of the floating cell.