Method for quantitative video-microscopy and associated system and computer software program product

A method of determining the amount of each of a plurality of molecular species comprising a sample, each molecular specie being indicated by a dye, at least one dye comprising a marker dye and another dye comprising a counterstain, said method comprising:

• determining an optical density of the sample from the image data, corresponding to an image of the sample captured by a color camera in a video-microscopy system, in each of a red, green, and blue channel and for a pixel in the image, so as to form a corresponding optical density matrix for the pixel;
• determining a relative absorption coefficient for at least the marker dye and the counterstain, independently of the sample and in each of the red, green, and blue channels, so as to form a corresponding relative absorption coefficient matrix;
• and
• multiplying the optical density matrix by an inverse of the relative absorption coefficient matrix so as to form a resultant matrix for the pixel, the resultant matrix comprising the amount of each molecular specie, as indicated by the respective dye, for the pixel.

A video-microscopy system for determining an amount of at least one molecular specie comprising a sample, each molecular specie being indicated by a dye, from an image of the sample, said system comprising:

• a color image acquisition device configured so as to be capable of capturing a magnified digital image of the sample as image data;
• a computer device operably engaged with the image acquisition device and comprising:
• a processing portion configured to determine an optical density of the sample from the image data, in each of a red, green, and blue channel and for a pixel in the image, so as to form a corresponding optical density matrix for the pixel;
• a processing portion configured to determine a relative absorption coefficient for the dye indicating each molecular specie. independently of the sample and in each of the red, green, and blue channels, so as to form a corresponding relative absorption coefficient matrix;
• and
• a processing portion configured to multiply the optical density matrix by an inverse of the relative absorption coefficient matrix so as to form a resultant matrix for the pixel, the resultant matrix comprising the amount of each molecular specie, as indicated by the respective dye, for the pixel.

A computer-readable medium encoded with a computer program capable of determining an amount of at least one molecular specie comprising a sample, each molecular specie being indicated by a dye, said computer-readable medium encoded with a computer program being executable on a computer device and comprising:

• an executable portion capable of determining an optical density of the sample from the image data, corresponding to a digital image of the sample captured by a color image acquisition device in a video-microscopy system, in each of a red, green, and blue channel and for a pixel in the digital image, so as to form a corresponding optical density matrix for the pixel;
• an executable portion capable of determining a relative absorption coefficient for the dye indicating each molecular specie, independently of the sample and in each of the red, green, and blue channels, so as to form a corresponding relative absorption coefficient matrix;
• and
• an executable portion capable of multiplying the optical density matrix by an inverse of the relative absorption coefficient matrix so as to form a resultant matrix for the pixel, the resultant matrix comprising the amount of each molecular specie, as indicated by the respective dye, for the pixel.

A method of determining an amount of at least one molecular specie comprising a sample, each molecular specie being indicated by a dye, said method comprising:

• determining an optical density of the sample, from an image of the sample captured by an RGB camera in a video-microscopy system, in each of a red, green, and blue channel of the RGR camera and for a pixel in the image, so as to form a corresponding optical density matrix for the pixel;
• determining a relative absorption coefficient for the dye indicating each molecular specie, independently of the sample and in each of the red, green, and blue channels, so as to form a corresponding relative absorption coefficient matrix;
• and
• multiplying the optical density matrix by an inverse of the relative absorption coefficient matrix so as to form a resultant matrix for the pixel, the resultant matrix comprising the amount of each molecular specie, as indicated by the respective dye, for the pixel.

A video-microscopy system for determining an amount of at least one molecular specie comprising a sample, each molecular specie being indicated by a dye, from an image of the sample, said system comprising:

• a microscope configured to form a magnified image of the sample;
• an RGB camera having a red channel, a green channel, and a blue channel, and operably engaged with tho microscope so as to he capable of forming a digital image from the magnified image;
• a Computer device operably engaged with the RGB camera and comprising:
• a processing portion configured to determine an optical density of the sample in each of the red, green, and blue channels and for a pixel in the digital image so as to form a corresponding optical density matrix for the pixel;
• a processing portion configured to determine a relative absorption coefficient for The dye indicating each molecular specie, independently of the sample and in each or the red, green, and blue channels, so as to form a corresponding relative absorption coefficient matrix;
• and
• a processing portion configured to multiply the optical density matrix by an inverse of the relative absorption coefficient matrix so as to form a resultant matrix for the pixel of the digital image, the resultant matrix comprising tho amount of each molecular specie, as indicated by the respective dye, for the pixel.

A computer-readable medium, encoded with a computer program capable of determining an amount of at least one molecular specie comprising a sample, each molecular specie being indicated by a dye, said computer-readable medium encoded with a computer program being configured to be executed on a computer device and comprising:

• an executable portion capable of determining an optical density of the sample, from a digital image of the sample captured by an RGB camera in a video-microscopy system, in each of the red, green, and blue channels of the RGB camera and for a pixel in the digital images so as to form a corresponding optical density matrix for the pixel;
• an executable portion capable of determining a relative absorption coefficient for the dye indicating each molecular specie, independently of the sample and in each of the red, green, and blue channels, so as to form a corresponding relative absorption coefficient matrix;
• and
• an executable portion capable of multiplying the optical density matrix by an inverse of the relative absorption coefficient matrix so as to form a resultant matrix for the pixel of the digital image, the resultant matrix comprising the amount of each molecular specie, as indicated by the respective dye, for the pixel.