Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 61

F

and the fluorescence quantum yield f ; thus preferably fluorophores should

have both high absorbance and fluorescence quantum yield. They should
possess a Stokes shift (i.e., spacing between excitation and emission wave-
lengths) large enough to avoid detection of scattered light from the excita-
tion beam. They must retain their bright fluorescence in the biological
milieu, so, ideally, fluorophores should be soluble in water and should
not aggregate in aqueous solution, or at least one should be able to formulate
them in a form in which they stay fluorescent in the biological environment.
It is also important that fluorophores should not interact (or should not
change their optical characteristic upon interaction) with biomolecules, par-
ticularly serum proteins. High chemo- and photostability and lack of (photo)
toxicity are other important characteristics of fluorophores. In addition,
fluorophores should be synthetically available, easy to functionalize, and able
to be attached to biomolecules or targeting/recognition units.
For a chemist, development of fluorophores for in vivo imaging is a par-
ticular challenge, as integration of all required attributes in one molecular
framework is highly demanding, if at all possible. Rational design of optimal
fluorophores starts from the selection of a molecular platform with suitable
optical properties and then requires gaining a deep insight into their
structure-property relationship in order to fine-tune their optical character-
istics. The next step is usually optimization of their chemical properties, such
as water solubility, bioconjugation, attaching targeting or recognition moi-
ety, and necessary chemical modification to improve their performance
in vivo (such as proper blood circulation, cell permeability, accumulation
in the target tissue, etc., depending on the desired application). The design
of efficient methods for synthesis and chemical modification of the target
systems is also a key part of fluorophore development. The lack of robust
synthetic methods is often the limiting factor in determining the
structure-property relationship and optimizing the properties of certain
fluorophores. The whole process of fluorophore development requires deep
insight into the electronic structure of the given molecules, robust synthetic
methods for their preparation and functionalization, and understanding the
biological processes that fluorophores undergo in vivo , thus requiring exper-
tise from various fields such as physics, chemistry, biochemistry, and biology.
There are several classes of fluorophores that have already been used or
can be potentially applied, in vivo , including small organic and inorganic
molecules, fluorescent proteins, conjugated polymers, and inorganic
nanoparticles. There are excellent up-to-date reviews covering fluorophores
for biological applications, 6,7

fluorescent molecular probes for biomedical