Detection method
A detection method includes: preparing sample image data of a sample in which a target substance is labeled with a labeling substance; acquiring first background image data in which an image feature amount of a single bright spot in the sample image data is reduced by a first filter; acquiring second background image data in which an image feature amount of a dense bright spot in the sample image data is reduced by a second filter; synthesizing the first background image data and the second background image data on a basis of brightness information of the sample image data and acquiring synthesized background image data; and obtaining difference image data that is a difference between the sample image data and the synthesized background image data.
1 . A detection method comprising:
preparing sample image data of a sample in which a target substance is labeled with a labeling substance;
acquiring first background image data in which an image feature amount of a single bright spot in the sample image data is reduced by a first filter;
acquiring second background image data in which an image feature amount of a dense bright spot in the sample image data is reduced by a second filter;
synthesizing the first background image data and the second background image data on a basis of brightness information of the sample image data and acquiring synthesized background image data;
obtaining difference image data that is a difference between the sample image data and the synthesized background image data; and
the image feature amount is an intensity distribution of light,
in the acquiring of the synthesized background image data,
an intensity difference that is a difference between a second intensity distribution of light in the second background image data and a first intensity distribution of light in the first background image data is obtained, and
a synthesized intensity distribution is acquired by using the first background image data in an area where the intensity difference is less than a first threshold value and using the second background image data in an area where the intensity difference is greater than or equal to the first threshold value.
2 . The detection method according to claim 1 , further comprising quantifying the target substance on a basis of the difference image data.
3 . The detection method according to claim 1 , wherein
the image feature amount is an intensity distribution of light, and
an intensity distribution of light in the sample image data reduced by the first filter and an intensity distribution of light in the sample image data reduced by the second filter are different in a peak width in the intensity distribution, a peak height in the intensity distribution, or the peak width and the peak height.
4 . The detection method according to claim 1 , wherein at least one of the first filter or the second filter is a median filter.
5 . The detection method according to claim 1 , wherein at least one of the first filter or the second filter is a frequency filter.
6 . The detection method according to claim 1 , wherein in the obtaining of the difference image data, noise tolerance processing of excluding the intensity distribution less than a second threshold value is further performed.
7 . The detection method according to claim 2 , wherein in the quantifying of the target substance, expansion processing is further performed.
8 . The detection method according to claim 6 , wherein
a plurality of sample image data obtained by synthesizing a plurality of first image data of a sample including only a labeling substance arranged at different densities and second image data of a sample acquired without labeling a target substance with the labeling substance is prepared,
an image feature amount of a bright spot is reduced by using the first filter and the second filter for each of the plurality of sample image data, and a plurality of reduced image data from which the bright spot is extracted is acquired by using the first threshold value and the second threshold value,
the difference image data that is a difference between each of the plurality of first image data and each of the plurality of reduced image data is acquired,
the plurality of first image data and a plurality of the difference image data are compared to each other, and
the first threshold value and the second threshold value are selected on a basis of a matching rate between the bright spot in the plurality of first image data and the bright spot in the plurality of the difference image data.