Fluorescence microscopy plays an important role in the classification of cancerous Tissue. The dramatic increase in multicolor fluorescence microscopy applications witnessed over the past decade is due, in part, to the significant advances in instrument and detector design. A number of advanced microscopy techniques have been applied using multi-color fluorescence labeling, including fluorescence recovery after photo bleaching (FRAP), fluorescence correlation spectroscopy (FCS), fluorescence resonance energy transfer (FRET), fluorescence in situ hybridization (FISH), and fluorescence lifetime imaging (FLIM). Many of these methods benefit significantly from the ability to use specifically targeted fluorescent proteins in live-cell imaging experiments. In addition, live-cell imaging has been revolutionized by the introduction of ever increasingly useful genetically encoded fluorescent proteins spanning the entire visible spectral region. However, the problem of fluorescence microscopy is the crosstalk between the channels caused by the overlap of the emission spectra of the different fluorophores, The crosstalk cannot be solved on the filter level, and not by specialized florophores. To eliminate the crosstalk the hyperspectral imaging using the spectra unmixing (algorithmically reduce the overlap of spectra) can be the possible way to reduce the errors in the classification of the tissue. Spectral imaging is the combination of commuter vision and spectroscopy. In addition, because every object of interest consists of more than one pixels, every pixel is dependent on its neighboring pixels. Thus, the spatial context of the image contains useful information for a classification and increase the sensitivity and specificity of a spectral classification.
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