A method of optically quantifying expression of at least one target molecule in at least one region of interest of a biological specimen, such as a tissue sample, comprising: a) producing a predetermined fractional sub-population of a total population of target molecules in the specimen as optically recognizable dots, wherein the predetermined fractional sub-population is equal or less than 99,9% of the total quantity of single molecules of the target in the sample, and wherein the dots are produced by using at least one of: 1) immunohistochemical target detection system, 2) non-antibody-based target detection system, and 3) enzyme- labeled molecular probe target detection system; b) imaging the specimen; c) selecting at least one region of interest within the image; d) recognizing at least one dot within the at least one region of interest; e) quantifying the dots within the at least one region of interest, wherein the step of quantifying the dots includes at least one: 1) counting the dots, including counting clusters of the dots, 2) measuring a total area of the dots, and 3) calculating a density of the dots in a unit area and f) assessing the expression of target molecules within the at least one region of interest based on the quantification of the dots; wherein (i) the features of size and shape of the optically recognizable dots and at least one additional optical feature selected from fluorescence, hue, diffraction level, sharpness, intensity and saturation are programmed in situ by intentional variation of conditions of chemical reaction producing the dots; and (ii) the number of the produced dots is representative of a predetermined fractional sub-population of a total population of target molecules within that region. a) producing a predetermined fractional sub-population of a total population of target molecules in the specimen as optically recognizable dots, wherein the predetermined fractional sub-population is equal or less than 99,9% of the total quantity of single molecules of the target in the sample, and wherein the dots are produced by using at least one of: 1) immunohistochemical target detection system, 2) non-antibody-based target detection system, and 3) enzyme- labeled molecular probe target detection system; 1) immunohistochemical target detection system, 2) non-antibody-based target detection system, and 3) enzyme- labeled molecular probe target detection system; b) imaging the specimen; c) selecting at least one region of interest within the image; d) recognizing at least one dot within the at least one region of interest; e) quantifying the dots within the at least one region of interest, wherein the step of quantifying the dots includes at least one: 1) counting the dots, including counting clusters of the dots, 2) measuring a total area of the dots, and 3) calculating a density of the dots in a unit area and 1) counting the dots, including counting clusters of the dots, 2) measuring a total area of the dots, and 3) calculating a density of the dots in a unit area and f) assessing the expression of target molecules within the at least one region of interest based on the quantification of the dots; (i) the features of size and shape of the optically recognizable dots and at least one additional optical feature selected from fluorescence, hue, diffraction level, sharpness, intensity and saturation are programmed in situ by intentional variation of conditions of chemical reaction producing the dots; and (ii) the number of the produced dots is representative of a predetermined fractional sub-population of a total population of target molecules within that region.