US-12618829-B2 - Method of analyzing neurons, device for analyzing neurons, and computer program

US12618829B2US 12618829 B2US12618829 B2US 12618829B2US-12618829-B2

Abstract

A method of analyzing neurons includes: identifying a cell region by identifying a region of a neurite or a region of a cell body of a neuron on the basis of time-series images obtained by imaging the neuron in time series; detecting aggregates by imaging, in time series, first fluorescent protein tagged to specific protein expressed in the neuron and detecting presence or absence of aggregates of the specific protein aggregated in the region of the neurite or the region of the cell body identified in the identifying the cell region on the basis of luminance of the first fluorescent protein included in the time-series images; and performing an analysis by classifying the neuron into a plurality of groups on the basis of a detection result of the detecting the aggregates and analyzing a survival state of the neuron for each of the groups.

Inventors

Toru Ichihashi
Takahiko Yoshida
Moeka NOBUTA

Assignees

NIKON CORPORATION

Dates

Publication Date: 20260505
Application Date: 20230711
Priority Date: 20210126

Claims (15)

1 . A method of analyzing neurons, comprising: acquiring an image by acquiring time-series images obtained by imaging a neuron in time series; identifying a cell region by identifying a region of a neurite or a region of a cell body of the neuron on a basis of the time-series images; detecting aggregates by detecting, on a basis of the time-series images obtained by imaging, in time series, first fluorescent protein tagged to specific protein expressed in the neuron, presence or absence of aggregates of the specific protein aggregated in the region of the neurite or the region of the cell body identified in the identifying the cell region by using, as a basis, luminance of the first fluorescent protein included in the time-series images; and performing an analysis by classifying the neuron into a plurality of groups on a basis of a detection result of the detecting the aggregates and analyzing a survival state of the neuron for each of the groups, wherein the survival state of the neuron includes at least any one of a survival period of time or a survival rate of the neuron, and the performing the analysis includes displaying, for each of the groups, a relationship between the plurality of groups and survival states of the plurality of groups.
2 . The method of analyzing neurons according to claim 1 , wherein the time-series images have at least one of data of a phase difference image, data of a fluorescence image including the luminance of the first fluorescent protein, or data including a moving image file.
3 . The method of analyzing neurons according to claim 2 , wherein the identifying the cell region includes imaging, in time series, the first fluorescent protein tagged to the specific protein expressed in the neuron in the acquiring the image, and identifying, as the region of the neurite of the neuron, a region where the luminance of the first fluorescent protein in the time-series images exceeds a first luminance threshold.
4 . The method of analyzing neurons according to claim 3 , wherein in the identifying the cell region, a region exceeding a predetermined size threshold among regions including the region where the luminance of the first fluorescent protein in the time-series images exceeds the first luminance threshold is identified as the region of the neurite or the cell body of the neuron, and further, each of the neurite being identified and identical and the cell body being identified and identical is tracked in time series, and a survival period of time related to the survival state is calculated.
5 . The method of analyzing neurons according to claim 3 , wherein the detecting the aggregates includes detecting the region of the neurite identified in the identifying the cell region as a region where the aggregates of the specific protein exist, and recognizing, as one aggregate region, a plurality of the aggregates in which a distance between regions where the aggregates exist is closer than a distance threshold in the time-series images.
6 . The method of analyzing neurons according to claim 3 , wherein in the identifying the cell region, a region where the luminance of the first fluorescent protein in the time-series images exceeds a second luminance threshold different from the first luminance threshold is identified as the region of the cell body of the neuron, and in the detecting the aggregates, the presence or absence of the aggregates of the specific protein is detected on a basis of comparison between the luminance of the first fluorescent protein and the first luminance threshold, in the region of the cell body identified in the identifying the cell region.
7 . The method of analyzing neurons according to claim 1 , wherein in the identifying the cell region, the region of the cell body or the neurite of the neuron is further identified on a basis of the time-series images obtained by imaging, in time series, second fluorescent protein expressed in the neuron and different from the first fluorescent protein, in the detecting the aggregates, whether the aggregates in the neurite identified exist in an axon or whether the aggregates exist in a dendrite is detected on a basis of the first fluorescent protein and the second fluorescent protein, and in the performing the analysis, on a basis of a detection result of the detecting the aggregates, the neuron is classified into a cell group in which the aggregates in the neurite exist on a side close to the cell body and a cell group in which the aggregates exist on a side close to a terminal of the neurite, and the survival state of the neuron is analyzed for each of the cell groups.
8 . The method of analyzing neurons according to claim 7 , further comprising performing association by specifying association between the cell body and the neurite constituting the neuron on a basis of the time-series images of the first fluorescent protein or the second fluorescent protein, wherein the performing the analysis includes classifying the neuron into a plurality of cell groups on a basis of the detection result of the presence or absence of the aggregates in the neurite and the presence or absence of the aggregates in the cell body in the detecting the aggregates with respect to the cell body and the neurite associated in the performing the association, and analyzing the survival state of the neuron for each of the cell groups.
9 . The method of analyzing neurons according to claim 3 , wherein the performing the analysis includes determining, on a basis of the luminance of the first fluorescent protein included in the time-series images, that the neuron is dead if luminance of the neurite or the cell body of the neuron is less than the first luminance threshold or less than a second luminance threshold, and calculating, as a survival period of time of the neuron, a period of time until the luminance of the neurite or the cell body is in a state of less than the first luminance threshold or less than the second luminance threshold, and displaying, on a display unit, a relationship between the detection result of the presence or absence of the aggregates in the neurite and the presence or absence of the aggregates in the cell body detected in the detecting the aggregates and the survival period of time of the neuron.
10 . A device for analyzing neurons, comprising: an imaging device that comprises a microscope and a camera and that acquires time-series images obtained by imaging a neuron in time series; and a processor programmed to: identify a region of a neurite or a region of a cell body of the neuron on a basis of the time-series images; detect, on a basis of the time-series images obtained by imaging, in time series, first fluorescent protein tagged to specific protein expressed in the neuron, presence or absence of aggregates of the specific protein aggregated in the identified region of the neurite or the region of the cell body of the neuron by using, as a basis, luminance of the first fluorescent protein included in the time-series images; and classify the neuron into a plurality of groups on a basis of a result of the detection and analyze a survival state of the neuron for each of the groups, wherein the survival state of the neuron includes at least any one of a survival period of time or a survival rate of the neuron, and the processor is further programmed to cause to be displayed, for each of the groups, a relationship between the plurality of groups and survival states of the plurality of groups.
11 . The device for analyzing neurons according to claim 10 , wherein the processor is further programmed to image, in time series, the first fluorescent protein tagged to the specific protein expressed in the neuron in the imaging device, and identify, as the region of the neurite of the neuron, a region where the luminance of the first fluorescent protein in the time-series images exceeds a first luminance threshold.
12 . The device for analyzing neurons according to claim 11 , wherein the processor is further programmed to identify, as the region of the neurite or the cell body of the neuron, a region exceeding a predetermined size threshold among regions including the region where the luminance of the first fluorescent protein in the time-series images exceeds the first luminance threshold, and further track, in time series, each of the neurite being identified and identical and the cell body being identified and identical, and calculate a survival period of time related to the survival state.
13 . The device for analyzing neurons according to claim 11 , wherein the processor is further programmed to detect the identified region of the neurite as a region where the aggregates of the specific protein exist, and recognize, as one aggregate region, a plurality of the aggregates in which a distance between regions where the aggregates exist is closer than a distance threshold in the time-series images.
14 . The device for analyzing neurons according to claim 11 , wherein the processor is further programmed to identify, as the region of the cell body of the neuron, a region where the luminance of the first fluorescent protein in the time-series images exceeds a second luminance threshold different from the first luminance threshold, and detect, in the region of the cell body identified, the presence or absence of the aggregates of the specific protein on a basis of comparison between the luminance of the first fluorescent protein and the first luminance threshold.
15 . A non-transitory computer-readable storage medium, which has stored thereon a computer program, having therein instructions that, when executed by a processor or a programmable circuit, cause the processor or the programmable circuit to perform operations comprising the method of analyzing neurons according to claim 1 .

Description

This application is a continuation application, filed under 35 U.S.C. § 111(a) of International Patent Application No. PCT/JP2022/001618 filed on Jan. 18, 2022, which claims priority benefit from Japanese Patent Application No. 2021-010409 filed on Jan. 26, 2021, the contents of each of which are incorporated herein by reference in their entireties. BACKGROUND 1. Technical Field The present invention relates to a method of analyzing neurons, a device for analyzing neurons, and a computer program. 2. Related Art Fluorescence observation of cells has been performed by introducing a gene encoding fluorescent protein into a living cell. As the fluorescent protein, green fluorescent protein (GFP), red fluorescent protein (RFP), and the like are used. These types of fluorescent protein are used as markers indicating localization of genes in cells and protein in cells. Patent Document 1 describes a method of introducing a gene encoding fluorescent protein into a plurality of cells and synchronizing cell cycles by using, as an index, fluorescence emitted from the cells. There is a need for a new technique for analyzing cells by using the fluorescent protein. Patent Document 1: Japanese Patent Application Publication No. 2009-072155 GENERAL DISCLOSURE In a first aspect of the present invention, a method of analyzing neurons is provided. The method of analyzing neurons may include acquiring an image by acquiring time-series images obtained by imaging a neuron in time series. The method of analyzing neurons may include identifying a cell region by identifying a region of a neurite or a region of a cell body of the neuron on the basis of the time-series images. The method of analyzing neurons may include detecting aggregates by detecting, on the basis of the time-series images obtained by imaging, in time series, first fluorescent protein tagged to specific protein expressed in the neuron, presence or absence of aggregates of the specific protein aggregated in the region of the neurite or the region of the cell body identified in the identifying the cell region by using, as a basis, luminance of the first fluorescent protein included in the time-series images. The method of analyzing neurons may include performing an analysis by classifying the neuron into a plurality of groups on the basis of a detection result of the detecting the aggregates and analyzing a survival state of the neuron for each of the groups. The performing the analysis may include displaying, for each of the groups, a relationship between the plurality of groups and survival states of the plurality of groups. In a second aspect of the present invention, the time-series images may have at least one of data of a phase difference image, data of a fluorescence image including the luminance of the first fluorescent protein, or data including a moving image file. In a third aspect of the present invention, the identifying the cell region may include imaging, in time series, the first fluorescent protein tagged to the specific protein expressed in the neuron in the acquiring the image. The identifying the cell region may include identifying, as the region of the neurite of the neuron, a region where the luminance of the first fluorescent protein in the time-series images exceeds a first luminance threshold. In a fourth aspect of the present invention, the identifying the cell region may include identifying, as the region of the neurite or the cell body of the neuron, a region exceeding a predetermined size threshold among regions including the region where the luminance of the first fluorescent protein in the time-series images exceeds the first luminance threshold. In the identifying the cell region, each of the neurite being identified and identical and the cell body being identified and identical may be tracked in time series, and a survival period of time related to the survival state may be calculated. In a fifth aspect of the present invention, the detecting the aggregates may include detecting the region of the neurite identified in the identifying the cell region as a region where the aggregates of the specific protein exist. The detecting the aggregates may include recognizing, as one aggregate region, a plurality of the aggregates in which a distance between regions where the aggregates exist is closer than a distance threshold in the time-series images. In a sixth aspect of the present invention, in the identifying the cell region, a region where the luminance of the first fluorescent protein in the time-series images exceeds a second luminance threshold different from the first luminance threshold may be identified as the region of the cell body of the neuron. In the detecting the aggregates, the presence or absence of the aggregates of the specific protein may be detected on the basis of a comparison between the luminance of the first fluorescent protein and the first luminance threshold in the region of the cell body identified in the identif