Emission wavelength, chromophore number

Further evidence for this assignment can be derived from the dependence of the amplitude a2 connected with the 7.4 ns component on the number of chromo-phores and the dependence on the emission wavelength (Aflu), respectively. As reported in Table 1.3, this amplitude is 0.8% for m-C 1P2 and increases to 4% for m-C 1P4 at 2nu = 600 nm. This is reasonable as the probability of formation of the excimer-like entity increases as the number of chromophores in the den-drimer increases. By detecting at 2nu = 725 nm, a2 increases to 1.3% for m-C 1P2 and to 6.3% for m-Cl P4. The larger contribution of that component at longer emission wavelengths is also consistent with a red-shifted fluorescence from excimer-like entities. This suggests that a fraction of the molecules have a substitution pattern in which two of the PI chromophores are relatively close in space. 

Various alternatives to luc have received attention recently. Notably Renilla luciferase (7,31-33) as well as a number of red-shifted mutants of luc. Although the use of Renilla luciferase, which emits at 480 nm and requires a different substrate, may have utihty in two-color essays, strong absorption of the emission wavelength limits use for in vivo imaging. The development and use of red-shifted luc mutants is primarily aimed at reducing optical attenuation by tissue. However, given the bandpass filter effect of tissue chromophores (absorption of wavelengths <600 nm), the spectral components of even the native Iwc-mediate luminescence emitted in vivo are primarily in the red part of the spectrum (>600 nm) (34). 

A growing number of clinical studies have demonstrated that fluorescence spectroscopy can be used to distinguish normal and abnormal human tissues in vivo in the skin, head and neck, genito-urinary tract, gastrointestinal tract, breast and brain. It is well known that fluorescence intensity and lineshape are a function of both the excitation and emission wavelength in samples containing multiple chromophores, such as human tissue. Major fluorescence contributors are structural proteins, NADH, FAD, tryptophane and porphyrins. 

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