Dye-Specific Wavelength Offsets to Resolve Spectrally Overlapping and Co-Localized Two-Photon Induced Fluorescence

The fundamentally important fluorescent imaging has one major limitation, resolution of over three dyes. This limitation is in part due to the overlap of the broad emission profile of each of the emitters used in fluorescence detection. The overlapping emission contaminates each emitter’s detection channel, referred to as cross-talk. To reduce fluorescence cross-talk for two photon applications, we present an innovative Two-Photon–Dye-Specific Excitation–Emission Offset (TP-DSO) method. TP-DSO selectively detects each dye by synchronously scanning the excitation and emission wavelengths at defined wavelength offsets. This technique advances multicolor analysis significantly by resolving dyes with highly overlapping spectral profiles. We identified three benefits: reduced excitation spectral bandwidth, reduced emission cross-talk between colocalized emitters with closely overlapping fluorescence, and validated use of thin-film variable optical emission filters for tuning the bandwidth and center wavelength. TP-DSO will advance multicolor analysis for many applications.