Nanoparticles for Bioimaging: Analytical Characterization and Measurements - Nanotechnology for Biomedical Imaging and Diagnostics - page 176

a parameter largely independent of the fluorescence intensity and fluorophore
concentration [75] but is sensitive to a great variety of biological factors that include
polarity, pH, viscosity, and temperature [76, 77].
flT characterization of the nanoparticles typically involves measuring the life-
times of a free dye, the dye associated with the nanoparticles, and the dye in the
biological environment. The alteration of the lifetime suggests an alteration of the
environment. for example, the increase of the lifetime of ICg from 0.2 to 0.55 ns
indicated the encapsulation of the dye; a subsequent increase to 0.65 ns evidenced
the release of the dye into from the nanoparticles into the bloodstream [40]. other
examples include validation of dye integration into PfC nanoparticles [78],
evaluation of liposome stability and leakage [79], and in vivo nanoparticle biode-
gradability [80].

6.7.3.2 Fluorescence Anisotropy fluorescence anisotropy is used to measure
hydrodynamic size of the nanoparticle [81] in order to verify the conjugation
efficiency of the fluorophores to nanoparticles and is used as a quality control tool for
fluorescently labeled nanoparticles [82, 83]. This optical technique has been widely
utilized in biochemistry and drug discovery [84] and has recently became a tool in
biomedical nanoparticle research. In fluorescence anisotropy, the fluorophore is irra-
diated with linearly polarized light. The resultant fluorescence intensity is measured
through a polarization filter placed in front of the detector and is oriented either
parallel or perpendicular to the incident polarized light. fluorescent anisotropy r can
then be determined from the following equation [85]:

I −

+

I

ΙΙ ⊥

r =

I 2 I

ΙΙ ⊥

where Ι II is the intensity the fluorescence emission parallel to the vertically polarized
light and I ⊥ is the intensity the fluorescence emission perpendicular to the vertically
polarized light.
anisotropy directly relates to the rotational correlation time ( θ ) of the molecules,
which is proportional to the hydrodynamic volume V of the nanoparticle in solution
according to a well-known Perrin equation [86], thus connecting the measurable
value r with the size of the nanoparticle:

r
1 τθ θ

η V

RT

r =

o

; =

+ /

where r is anisotropy, r 0 is limiting anisotropy (maximum possible, usually 0.4), τ is
flT, θ is rotational correlation time of a macromolecule, η is viscosity in poise,
V is volume of macromolecule, R is gas constant = 8.31 × 10 7 erg/mol K, and T is
temperature, K.