Biological material quantifying method, image processing device, pathological diagnosis support system and recording medium
A method of quantifying a specific biological material in a specimen stained using fluorescent dye accumulating particles capable of binding to the material includes: inputting a first fluorescence image obtained by capturing an image of the specimen; extracting a certain region from the first fluorescence image and calculating a first luminance integrated value by integrating luminance values of the certain region; and calculating the number of the particles included in the certain region from the first luminance integrated value and an average luminance value per fluorescent dye accumulating particle, wherein the average luminance value is calculated from a distribution of second luminance integrated values obtained by integrating luminance values for individual bright spot regions, which indicate emission of light by the fluorescent dye accumulating particles, in a second fluorescence image obtained by capturing an image of a preparation onto which the fluorescent dye accumulating particles are dispersed without agglomerating.
1. A biological material quantifying method of quantifying a specific biological material in a specimen that has been stained using fluorescent dye accumulating particles capable of binding to the specific biological material, the biological material quantifying method comprising:
an input step of inputting a first fluorescence image obtained by capturing an image of the specimen;
a luminance calculation step of extracting a certain region from the first fluorescence image and calculating a first luminance integrated value by integrating luminance values of the certain region; and
a particle count calculation step of calculating the number of the fluorescent dye accumulating particles included in the certain region from the first luminance integrated value and an average luminance value per said fluorescent dye accumulating particle,
wherein the average luminance value is calculated from a distribution of second luminance integrated values obtained by integrating luminance values for individual bright spot regions, which indicate emission of light by the fluorescent dye accumulating particles, in a second fluorescence image obtained by capturing an image of a preparation onto which the fluorescent dye accumulating particles are dispersed without agglomerating.
2. The biological material quantifying method according to claim 1 , wherein a density of the fluorescent dye accumulating particles on the preparation is 10 6 counts/mm 2 or less.
3. The biological material quantifying method according to claim 1 , satisfying the following Formula (1):
0≤ Sσ/S ave <1.0, Formula (1):
wherein Sσ represents a standard deviation of areas of the bright spot regions and S ave represents an average value of areas of the bright spot regions.
4. The biological material quantifying method according to claim 1 , satisfying the following Formula (2):
0.10< S ave ·π( R ave /2+Δ) 2 <10.00, Formula (2):
wherein S ave represents an average value of areas of the bright spot regions, R ave represents an average particle size of the fluorescent dye accumulating particles, and Δ represents a resolution of an instrument that captures the fluorescence images.
5. The biological material quantifying method according to claim 1 , wherein the average luminance value is calculated using 10,000 or more luminance values for the individual bright spot regions.
6. The biological material quantifying method according to claim 1 , wherein an average particle size of the fluorescent dye accumulating particles is 20 to 200 nm.
7. The biological material quantifying method according to claim 1 , wherein a coefficient of variation of a particle size of the fluorescent dye accumulating particles is 15% or less.
8. The biological material quantifying method according to claim 1 , wherein the average luminance value is a mode of the second luminance integrated values.
9. The biological material quantifying method according to claim 1 , wherein the average luminance value is a maximum luminance value in a fitting curve or an interpolation curve that is created by performing fitting or interpolation to the distribution of the second luminance integrated values.
10. The biological material quantifying method according to claim 1 , wherein the average luminance value is an average luminance value of fluorescent dye accumulating particles with a given particle size, calculated by using a lookup table or an interpolation curve that is created from the distribution of the second luminance integrated values based on multiple types of fluorescent dye accumulating particles with different average particle sizes.
11. The biological material quantifying method according to claim 1 , wherein the average luminance value is an average luminance value at a fluorescence wavelength of a given particle size, calculated by using a lookup table or an interpolation curve that is created from the distribution of the second luminance integrated values based on multiple types of fluorescent dye accumulating particles with different emission wavelengths.
12. The biological material quantifying method according to claim 1 , comprising a calibration step of calibrating the average luminance value per particle when emission property of the fluorescent dye accumulating particles changes.
13. The biological material quantifying method according to claim 1 , comprising a preparation production step of producing the preparation by dispersing the fluorescent dye accumulating particles and dissolvable particles onto a slide glass and then removing said particles by dissolution.
14. The biological material quantifying method according to claim 1 , comprising a preparation production step of producing the preparation by dispersing the fluorescent dye accumulating particles onto a slide glass that has been subjected to patterning with a dissolvable resin and then removing said resin by dissolution.
15. An image processing device that quantifies a specific biological material in a specimen that has been stained using fluorescent dye accumulating particles capable of binding to the specific biological material, the image processing device comprising:
an input unit that inputs a first fluorescence image obtained by capturing an image of the specimen; and
a hardware processor that extracts a certain region from the first fluorescence image, calculates a first luminance integrated value by integrating luminance values of the certain region, and
calculates the number of the fluorescent dye accumulating particles included in the certain region from the first luminance integrated value and an average luminance value per said fluorescent dye accumulating particle,
wherein the average luminance value is calculated from a distribution of second luminance integrated values obtained by integrating luminance values for individual bright spot regions, which indicate emission of light by the fluorescent dye accumulating particles, in a second fluorescence image obtained by capturing an image of a preparation onto which the fluorescent dye accumulating particles are dispersed without agglomerating.
16. A pathological diagnosis support system comprising:
the image processing device according to claim 15 ; and
an image acquiring device that acquires the first fluorescence image and the second fluorescence image.
17. A non-temporary, computer-readable recording medium that stores a program, the program causing a computer that quantifies a specific biological material in a specimen that has been stained using fluorescent dye accumulating particles capable of binding to the specific biological material to:
extract a certain region from a first fluorescence image obtained by capturing an image of the specimen; calculate a first luminance integrated value by integrating luminance values of the certain region; and
calculate the number of the fluorescent dye accumulating particles included in the certain region from the first luminance integrated value and an average luminance value per said fluorescent dye accumulating particle,
wherein the average luminance value is calculated from a distribution of second luminance integrated values obtained by integrating luminance values for individual bright spot regions, which indicate emission of light by the fluorescent dye accumulating particles, in a second fluorescence image obtained by capturing an image of a preparation onto which the fluorescent dye accumulating particles are dispersed without agglomerating.