Methods apparatuses and systems for detecting and quantifying phosphoproteins

A method of screening a drug for effectiveness in treating leukemia, the method comprising:

• a) contacting a leukemia cell with the drug;
• b) contacting the leukemia cell with a quantum dot labeled antibody specific for phosphorylated Abl, Akt, Crkl, ERK, STAT3, or STAT5;
• c) localizing a set of quantum dots in a 3-dimensional quantum dot image stack of a sample using a computing device by the steps of:
• i) receiving the 3-dimensional quantum dot image stack of the sample, wherein the sample includes a cell and the stack comprises a plurality of slices;
• ii) generating a cumulative histogram of all pixel intensities greater than zero in the quantum dot image stack of the sample;
• iii) calculating a low threshold and a high threshold based on the cumulative histogram;
• iv) identifying a first pixel location of a candidate quantum dot in a first slice of the quantum dot image stack of the sample based on the low threshold and the high threshold;
• v) pairing the first pixel location of the candidate quantum dot with a second pixel location of the candidate quantum dot in a second slice of the quantum dot image stack of the sample, wherein the second slice is adjacent to the first slice and pairing the first or second pixel location of the first candidate quantum dot with a third pixel location of the candidate quantum dot in a third slice of the quantum dot image stack of the sample wherein the third slice is adjacent to either the first slice or the second slice, thereby creating a quantum dot string for the candidate quantum dot;
• vi) detecting a location, in the quantum dot image stack of the sample, of a membrane of the cell using a membrane staining image stack;
• vii) determining the positions of the first and second quantum dots relative to the location of the membrane;
• and
• viii) outputting a pixel location of the candidate quantum dot and a position of the candidate quantum dot relative to the membrane;
• d) counting the number of quantum dots per cell in a set of the cells using fluorescence microscopy;
• e) wherein a result showing that the average cell has less than 10 quantum dots per cell indicates that the drug is effective in treating leukemia.