Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 91

contain toxic metals, such as cadmium and selenium. 6 Recently, fluorescent
proteins with large Stokes shifts, named Keima, have been developed,
which, in combination with other fluorescent proteins, allow multicolor
intracellular imaging with single excitation wavelengths (simultaneous
imaging using six different fluorescent proteins has been
demonstrated). 226 The wavelengths of excitation and emission for Keima
( 620 nm for the longest emitting variant) make

them less suitable for in vivo applications.
So far, in vivo multicolor imaging has been pursued using a cocktail of
organic fluorophores (such as mixture of various rhodamines, coumarines,
cyanine dyes, and/or Alexa Fluors), 227-230 quantum dots (also together
with organic fluorophores), 231-235 or upconverting nanocrystals. 236,237
Upconverting nanocrystals 238 are a unique class of fluorophores that are
composed of, for example, sodium yttrium fluoride and are doped
with rare-earth metal cations and they can be excited in the near-IR
region (e.g., 980 nm) and emit at a shorter wavelength. The
wavelength of emission depends on their composition. Upconverting
nanocrystals have been utilized in vivo for multicolor imaging alone, 236
or as an energy-transfer donor in conjugation with near-IR organic
fluorophores (rhodamines). 237
Promising fluorophores for in vivo multicolor applications are hydro-
porphyrin energy-transfer dyads (see Section 5.5 and Chart 3.8 ). The intrin-
sic properties of hydroporphyrins, that is, narrow and tunable absorption and
emission bands, together with their tunable apparent Stokes shift, which can
be achieved by assembling hydroporphyrins in energy-transfer dyads, make
themwell suited for multicolor imaging. Holten and coworkers studied pairs
of model chlorin-bacteriochlorin dyads where each dyad had the same
bacteriochlorin acceptor (thus the same emission wavelength) and different
chlorin components (thus different excitation wavelengths). 202,239 Because
of the narrow absorption bands in chlorins, the 25-nm separation between
the absorption maxima of both chlorins in the studied pair is sufficient for
selective excitation one dyad in the presence of the other. This selectivity
has been demonstrated also in vivo 239 and gives the promise for the
development of a new class of fluorophores with either a common
emission wavelength and different, well-resolved absorption bands, or a
common excitation wavelength and different, well-resolved emission
wavelengths.
An elegant strategy for fluorophores for multicolor imaging has been
developed by Kool and coworkers. 240,241 They assembled deoxyriboside
monomers containing small

l ¼ 440 nm, l ¼ abs em

fluorescent organic molecules (aromatic