Identification of objects of interest using multiple illumination schemes and finding overlap of features in corresponding multiple images

A method of detecting particular cells in a sample region that contains additional objects, the method comprising:

• providing a sample stained with a first dye that preferentially stains the cytoplasm of the particular cells and a second dye that preferentially stains cell nuclei;
• trans-illuminating the sample with first light in a wavelength range that is absorbed by the first dye;
• simultaneously illuminating the sample with second light that causes the second dye to fluoresce and emit third light;
• forming a single image of the first light passing through the sample and the third light emitted by the sample;
• and
• processing the single image to identify cells that have both stained cytoplasm and nuclei.

A method of detecting fetal nucleated red blood cells in a sample region that contains additional objects, the method comprising:

• providing a sample stained with a first dye that preferentially stains cytoplasm and a second dye that preferentially stains cell nuclei;
• trans-illuminating the sample with first light in a wavelength range that is absorbed by the first dye;
• forming a first image of the first light passing through the sample;
• processing the first image to produce a cytoplasm contrast mask having regions representing the presence of cytoplasm;
• illuminating the sample with second light that causes the second dye to fluoresce and emit third light;
• forming a second image of the third light emitted by the sample;
• processing the second image to produce a nuclear contrast mask having regions representing the presence of cell nuclei;
• combining the cytoplasm contrast mask and the nuclear contrast mask to determine regions of connected ON pixels in the cytoplasm contrast mask that are sufficiently close to regions of connected ON pixels in the nuclear contrast mask to signify the likely presence of fetal nucleated red blood cells.

A method of detecting fetal nucleated red blood cells in a sample region that contains additional objects, the method comprising:

• providing a sample stained with a first dye that preferentially stains fetal cytoplasm and a second dye that preferentially stains cell nuclei;
• trans-illuminating the sample with first light in a wavelength range that is absorbed by the first dye;
• illuminating the sample with second light that causes the second dye to fluoresce and emit third light;
• forming a single image of the first light passing through the sample and the third light emitted by the sample;
• determining a background level for the single image;
• generating a cytoplasm image by increasing pixel values in the single image by a second amount so that background regions are generally at maximum intensity;
• thresholding the cytoplasm image to create a binary cytoplasm contrast mask having regions having ON pixels representing the presence of objects stained by the first dye;
• generating a nuclear image by reducing pixel values in the single image by a first amount so that background regions are generally at minimum intensity;
• thresholding the nuclear image to create a binary nuclear contrast mask having regions having ON pixels representing the presence of objects stained by the second dye;
• morphologically processing each mask to expand regions of connected ON pixels, thereby producing processed masks;
• logically ANDing the processed masks to produce an intersection image that defines regions of overlap or near overlap of objects in the cytoplasm image stained by the first dye and objects in the nuclear image stained by the second dye;
• morphologically processing the intersection image to expand regions of connected ON pixels, thereby producing a seed image;
• and
• reconstructing the seed image within the cytoplasm contrast mask and the nuclear contrast mask to represent cytoplasm and nuclei for those objects that are likely to be fetal nucleated red blood cells.

A method of finding objects of interest that are interspersed with other objects in a sample, the method comprising:

• providing a sample where objects have optical properties so that objects of interest, when imaged on an imaging medium under a plurality of different contrasts in an illumination scheme, exhibit a combination of features, referred to as the unique combination, that is different from combinations of features exhibited by other objects;
• subjecting the sample to the plurality of different contrasts;
• generating a corresponding plurality of images wherein the plurality of images are derived from a lesser number of images, wherein at least one of the lesser number of images is a pre-combined image formed from the functional combination or logical combination of images taken with a combination of different contrasts;
• and
• analyzing the plurality of images to determine instances where the unique combination of features from the different images meets a predetermined proximity constraint.

A method of detecting particular cells in a sample region that contains additional objects, the method comprising:

• providing a sample stained with a first dye that preferentially stains the cytoplasm of the particular cells and a second dye that preferentially stains cell nuclei;
• trans-illuminating the sample with first light in a wavelength range that is absorbed by the first dye to form a first image;
• trans-illuminating the sample with second light in a wavelengths range this is absorbed by the second dye to from a second image;
• forming a single image of the first and second light passing through the sample by linearly combining or logically combining the first image and the second image;
• and
• processing the single image to identify cells that have both stained cytoplasm and nuclei.

A method of detecting fetal nucleated red blood cells in a sample region that contains additional objects, the method comprising:

• providing a sample stained with a first dye that preferentially stains cytoplasm and a second dye that preferentially stains cell nuclei;
• trans-illuminating the sample with first light in a wavelength range that is absorbed by the first dye;
• forming a first image of the first light passing through the sample;
• processing the first image to produce a cytoplasm contrast mask having regions representing the presence of cytoplasm;
• trans-illuminating the sample with second light in a wavelength range that is absorbed by the second dye;
• forming a second image of the second light passing through the sample;
• processing the second image to produce a nuclear contrast mask having regions representing the presence of cell nuclei;
• combining the cytoplasm contrast mask and the nuclear contrast mask to determine regions of connected ON pixels in the cytoplasm contrast mask that are sufficiently close to regions of connected ON pixels in the nuclear contrast mask to signify the likely presence of fetal nucleated red blood cells.

A method of detecting fetal nucleated red blood cells in a sample region that contains additional objects, the method comprising:

• providing a sample stained with a first dye that preferentially stains fetal cytoplasm and a second dye that preferentially stains cell nuclei;
• trans-illuminating the sample with first light in a wavelength range that is absorbed by the first dye to form a first image;
• trans-illuminating the sample with second light in a wavelength range that is absorbed by the second dye to form a second image;
• forming a single image of the first light passing through the sample and the second light passing through the sample by linearly combining or logically combining the first image and the second image;
• determining a background level for the single image;
• generating a cytoplasm image by increasing pixel values in the single image by a second amount so that background regions are generally at maximum intensity;
• thresholding the cytoplasm image to create a binary cytoplasm contrast mask having regions having ON pixels representing the presence of objects stained by the first dye;
• generating a nuclear image by reducing pixel values in the single image by a first amount so that background regions are generally at minimum intensity;
• thresholding the nuclear image to create a binary nuclear contrast mask having regions having ON pixels representing the presence of objects stained by the second dye;
• morphologically processing each mask to expand regions of connected ON pixels, thereby producing processed masks;
• logically ANDing the processed masks to produce an intersection image that defines regions of overlap or near overlap of objects in the cytoplasm image stained by the first dye and objects in the nuclear image stained by the second dye;
• morphologically processing the intersection image to expand regions of connected ON pixels, thereby producing a seed image;
• and
• reconstructing the seed image within the cytoplasm contrast mask and the nuclear contrast mask to represent cytoplasm and nuclei for those objects that are likely to be fetal nucleated red blood cells.