A method, comprising: obtaining multispectral image information for a sample comprising a fluorescent dye, wherein the multispectral image information corresponds to an image cube comprising multiple two-dimensional layers, each layer corresponding to an image of the sample; calculating from the image cube a first spectrum comprising contributions from the fluorescent dye and from endogenous fluorescence in the sample; calculating from the image cube a second spectrum comprising contributions from the fluorescent dye and from endogenous fluorescence in the sample; and calculating a pure spectrum of the fluorescent dye in the sample based on the first and second spectra, wherein a relative contribution of light emission from the fluorescent dye to the second spectrum is larger than a relative contribution of light emission from the fluorescent dye to the first spectrum; wherein calculating the first and second spectra comprises identifying corresponding first and second sets of pixel intensity values in the image cube and using the identified sets of pixel intensity values to calculate the first and second spectra; wherein identifying the first set of pixel intensity values comprises designating one or more layers of the image cube as a first layer set, and identifying members of the first set of pixel intensity values based on the first layer set; and wherein calculating the pure spectrum comprises subtracting a selected amount of the first spectrum from the second spectrum where the selected amount is based on a priori knowledge about the fluorescent dye and the endogenous fluorescence in the sample, wherein the a priori knowledge includes knowledge about a dark band within the first and second spectra, which is a combination of excitation and emission wavelengths for which the dye is known to exhibit little or no fluorescence, and wherein the selected amount is determined based on a criterion imposing a low signal level of the pure spectrum in the dark band. obtaining multispectral image information for a sample comprising a fluorescent dye, wherein the multispectral image information corresponds to an image cube comprising multiple two-dimensional layers, each layer corresponding to an image of the sample; calculating from the image cube a first spectrum comprising contributions from the fluorescent dye and from endogenous fluorescence in the sample; calculating from the image cube a second spectrum comprising contributions from the fluorescent dye and from endogenous fluorescence in the sample; and calculating a pure spectrum of the fluorescent dye in the sample based on the first and second spectra, wherein a relative contribution of light emission from the fluorescent dye to the second spectrum is larger than a relative contribution of light emission from the fluorescent dye to the first spectrum; wherein calculating the first and second spectra comprises identifying corresponding first and second sets of pixel intensity values in the image cube and using the identified sets of pixel intensity values to calculate the first and second spectra; wherein identifying the first set of pixel intensity values comprises designating one or more layers of the image cube as a first layer set, and identifying members of the first set of pixel intensity values based on the first layer set; and wherein calculating the pure spectrum comprises subtracting a selected amount of the first spectrum from the second spectrum where the selected amount is based on a priori knowledge about the fluorescent dye and the endogenous fluorescence in the sample, wherein the a priori knowledge includes knowledge about a dark band within the first and second spectra, which is a combination of excitation and emission wavelengths for which the dye is known to exhibit little or no fluorescence, and wherein the selected amount is determined based on a criterion imposing a low signal level of the pure spectrum in the dark band. wherein a relative contribution of light emission from the fluorescent dye to the second spectrum is larger than a relative contribution of light emission from the fluorescent dye to the first spectrum; wherein calculating the first and second spectra comprises identifying corresponding first and second sets of pixel intensity values in the image cube and using the identified sets of pixel intensity values to calculate the first and second spectra; wherein identifying the first set of pixel intensity values comprises designating one or more layers of the image cube as a first layer set, and identifying members of the first set of pixel intensity values based on the first layer set; and wherein calculating the pure spectrum comprises subtracting a selected amount of the first spectrum from the second spectrum where the selected amount is based on a priori knowledge about the fluorescent dye and the endogenous fluorescence in the sample, wherein the a priori knowledge includes knowledge about a dark band within the first and second spectra, which is a combination of excitation and emission wavelengths for which the dye is known to exhibit little or no fluorescence, and wherein the selected amount is determined based on a criterion imposing a low signal level of the pure spectrum in the dark band.