Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 60

The recent designing and synthetic efforts have been dedicated to improving their op-
tical properties (shift the absorption and emission maxima toward longer wavelengths
and increase the brightness) as well as increasing their stability and water solubility. The
most notable advances include development of encapsulated cyanine dyes with in-
creased stability and water solubility, squaraine rotaxanes with increased stability,
long-wavelength-absorbing boron dipyrromethenes, long-wavelength-absorbing por-
phyrin and hydroporphyrin derivatives, and water-soluble phthalocyanines. Recent ad-
vances in luminescence and bioluminescence have made self-illuminating fluorophores
available for applications. Development of new types of hydroporphyrin energy-

transfer dyads gives the promise for further advances in in vivo multicolor imaging.

in vivo

1. INTRODUCTION
Fluorescence spectroscopy, which has been proved to be an extremely
powerful tool in analytical chemistry and cell biology, 1,2 has reached the
stage when it can be used to visualize biological processes and detect
biologically relevant species in tissues and in whole living animals. It
opens a particularly fascinating opportunity to noninvasively diagnose
disease stages at the molecular level, which can revolutionize medicinal
diagnosis. The progress in applications of fluorescence spectroscopy for in
vivo imaging relies on both advances in excitation and detection
technologies and development of molecular probes suitable for
visualization of molecular processes in tissue or whole body. The
fluorescent molecular probes must consist of two components: a reporter
(fluorophore), that is, the unit that emits the light upon excitation, and a
recognition unit, which can selectively recognize the given molecular
process or species of interest and translate the recognition event in a well-
defined manner into changes of the fluorescence properties of a
reporter. 1,2 Thus, fluorophore as a reporter is a centerpiece of every
fluorescence molecular probe. Fluorophores for in vivo applications must
fulfill a set of requirements as for their optical, chemical, and biological
properties. The most critical properties are summarized below. 3-5
For in vivo applications, fluorophores must absorb and emit in the red and
near-infrared (near-IR) spectral window, preferably in the range
650-900 nm. In this window, the tissue absorbance and autofluorescence,
as well as light scattering, are diminished, while below 650 nm, tissue and
cellular components strongly absorb the light and above 900 nm water ab-
sorbs (e.g., see discussion in Ref. 5 ). Fluorophores should possess high
brightness, which is a product of the fluorophore excitation coefficient

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