Excitation unmixing

Excitation-based unmixing is being used in commercially available setups for widefield as well as for two-photon imaging. In contrast to the parallel detection possibility for emission unmixing, excitation unmixing is unavoidably based on sequentially acquired data. 

Thaler, C. and Vogel, S. S. (2006). Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation. Cytometry A 69, 904—11. 

Above it was pointed out that in unmixed rare gases binary absorption does not exist because of the inversion symmetry of like pairs. For like molecular pairs inversion symmetry does in general not exist because of the anisotropic structure and vibrational excitations of the individual molecules. In Chapter 5, we will show that in pure hydrogen gas, for example, the translational spectrum arises mainly from orientational ( magnetic ) transitions the translational spectrum of H2-H2 is discernible in Fig. 3.10 at low frequencies (0 < v < 250 cm-1). The translational peak is weak if compared to the strong So(J) lines near 354 and 587 cm-1, but its strength is comparable to those of the dissimilar rare gas pairs, Fig. 3.1. The translational H2-He pair spectra are somewhat stronger, Fig. 3.12,... 

We note that even the unmixed molecular gases absorb infrared radiation by collisional induction, in contrast to the unmixed monatomic gases which do not. This is so because pairs of like atoms possess an inversion symmetry that is inconsistent with a dipole moment pairs of like molecules, on the other hand, may be found in various orientations and rotovibrational excitations that are consistent with a dipole moment of the complex. We will, therefore, consider in this Chapter mostly the unmixed molecular gases (without neglecting mixtures unduly). 

As the number of fluorophores separable by Unear unmixing is limited by the number of channels available for analysis (see Table l),the combination of excitation and emission unmixing even increases the number of fluorophores that can be distinguished in a sample. 

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