Principles of Fluorescence Spectroscopy

70 FLUOROPHORES
Figure 3.11. Structures and normalized fluorescence emission spectra
of goat anti-mouse IgG conjugates of (1) fluorescein, (2) rhodamine
6G, (3) tetramethylrhodamine, (4) Lissamine rhodamine B, and (5)
Texas Red dyes. Revised from [30].
rescein and rhodamine. The BODIPY dyes have a small
Stokes shift and usually display self-quenching. New dyes
are being developed that show both a large Stokes shift and
good water solubility. One such dye is Cascade Yellow,
which displays excitation and emission maximum near 409
and 558 nm, respectively (Figure 3.9). The large Stokes
shift minimizes the tendency for homotransfer, and the
charges on the aromatic rings aid solubility.
In contrast to fluorescein, rhodamines, and BODIPYs,
there are fluorophores that display high sensitivity to the
polarity of the local environment. One example is Prodan 35
(Figure 3.8), which is available in the reactive form—called
Figure 3.12. Normalized fluorescence emission spectra of BODIPY
fluorophores in methanol. Revised from [30].
acrylodan. 36 In the excited state there is a charge separation
from the amino to the carbonyl groups. When bound to
membranes, Prodan and its derivatives display large spectral
shifts at the membrane phase-transition temperature.
3.2.3. Photostability of Fluorophores
One of the most important properties of a probe is its photostability.
Almost all fluorophores are photobleached upon
continuous illumination, especially in fluorescence
microscopy where the light intensities are high. Fluorescein
is one of the least photostable dyes (Figure 3.14). The Alexa