A method of analyzing a sample, the sample comprising a first detection moiety having a first emission spectrum, the first emission spectrum having a peak emission intensity at a peak intensity wavelength, a leading spectral edge with emission intensities less than the peak intensity at wavelengths less than the peak intensity wavelength, and a trailing spectral edge with emission intensities less than the peak intensity at wavelengths greater than the peak intensity wavelength, the method comprising: acquiring, with an imaging device by stimulating the sample with excitation light, a first set of raw images, the first set of raw images consisting of: a first raw image comprising a first emission signal while stimulating the sample with the excitation light, wherein the first emission signal comprises an intensity at a first emission wavelength of the first detection moiety, and a second raw image comprising a second emission signal while stimulating the sample with the excitation light, wherein the second emission signal comprises an intensity at a second emission wavelength of the first detection moiety, wherein the intensity of the first emission signal is less than the intensity of the second emission signal within the first emission spectrum; calculating a slope of a percentage change in signal intensity between pixels of the first raw image and the corresponding pixels of the second raw image, the slope between the respective pixels defined as the percentage change between the intensity of the first emission signal and the intensity of the second emission signal divided by the change in the first emission wavelength and the second emission wavelength; wherein if the calculated slope is at least plus or minus (+/-) 5%/nm, then the pixels of the first raw image and the pixels of the second raw image are attributed to the first detection moiety; and outputting, with a processor, a final image of the first detection moiety based on the first and second raw images. acquiring, with an imaging device by stimulating the sample with excitation light, a first set of raw images, the first set of raw images consisting of: a first raw image comprising a first emission signal while stimulating the sample with the excitation light, wherein the first emission signal comprises an intensity at a first emission wavelength of the first detection moiety, and a second raw image comprising a second emission signal while stimulating the sample with the excitation light, wherein the second emission signal comprises an intensity at a second emission wavelength of the first detection moiety, wherein the intensity of the first emission signal is less than the intensity of the second emission signal within the first emission spectrum; a first raw image comprising a first emission signal while stimulating the sample with the excitation light, wherein the first emission signal comprises an intensity at a first emission wavelength of the first detection moiety, and a second raw image comprising a second emission signal while stimulating the sample with the excitation light, wherein the second emission signal comprises an intensity at a second emission wavelength of the first detection moiety, wherein the intensity of the first emission signal is less than the intensity of the second emission signal within the first emission spectrum; calculating a slope of a percentage change in signal intensity between pixels of the first raw image and the corresponding pixels of the second raw image, the slope between the respective pixels defined as the percentage change between the intensity of the first emission signal and the intensity of the second emission signal divided by the change in the first emission wavelength and the second emission wavelength; wherein if the calculated slope is at least plus or minus (+/-) 5%/nm, then the pixels of the first raw image and the pixels of the second raw image are attributed to the first detection moiety; and outputting, with a processor, a final image of the first detection moiety based on the first and second raw images.