Autofluorescence emission spectra

The quantification of fluorescent particles in cellular systems is difficult because several aspects such as autofluorescence, bleaching (see below), and quenching hamper analysis. Keep in mind that many fluorophores show a pH-dependent change in emission spectrum and intensity fluorescein-labeled dextrans (FITC-dextran) and calcein are strongly quenched upon acidification. If available, one should read the fluorescence intensity at its isosbestic point, where the intensity is not pH dependent. 

Yellow autofluorescence of lignin in UV-light (40,41) has also been used as a criterion (28,30,31). However, in view of the countless autofluorescent substances in plants (40,42), even the recording of a typical emission spectrum for lignin (31) cannot be regarded as a specific proof for the presence of lignin. 

Beardmore et al (30) and Tiburzy (31) showed that epidermal (31) and mesophyll (30,31) cells of resistant wheat plants, penetrated by haus-toria of an avirulent race of the fungus, can be stained with phlorogluci-nol/HCl (31) and chlorine/sulfite (30,31). These cells show yellow autofluorescence under UV-light (30,31), the emission spectrum is identical to that of lignified tracheary elements (31). 

Up to this point, it is clear that high-contrast (autofluorescence-free) NIR in vivo imaging requires the use of luminescent materials that show intense fluorescence in the NIR-IIa region. The emission spectrum of Nd (after 808 nm excitation) displays three emission bands centered at around 900,1060, and 1340 nm corresponding respectively to the F3/2 %i2,... 

Rhodamine is a preferred fluorochrome over fluorescein because of its slower bleach rate and its emission in a spectrum that shows less cellular autofluorescence. Also, this spectrum produces less autofluorescence in plastic substrata (see Chapter 14). Rhodamine requires a mercury vapor light source, since other sources, such as xenon, do not have sufficient emission in the green spectrum. 

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