Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels

Studies of biological samples often call for simultaneous identification of multiple molecular or structural components. Multiple labelling fluorescence techniques are a powerful way of achieving this. However, the ability to distinguish a number of fluorescent probes unambiguously can be restricted by the fact that fluorescence spectra are generally broad and overlapping. Recently a technique known as linear unmixing has been combined with spectral imaging to discriminate between multiple fluorophores. In this study a scheme is proposed whereby fluorescence polarization information is used to expand the capability of the linear unmixing technique to accommodate additional fluorescent probes. As a proof-of-concept, it is shown that this polarization-based technique can be used to divide the signals generated by two spectrally similar fluorescent probes into their separate components.