Nanoparticles for Bioimaging: Analytical Characterization and Measurements - Nanotechnology for Biomedical Imaging and Diagnostics - page 174

6.7.2.2 Fluorescence Quantum Yield—Relative Method fluorescence quantum
yields of the nanoparticles carrying organic fluorophores are measured the same way
as individual fluorophores using either relative or absolute measurements. The
relative method relies on a reference dye that already has a defined quantum yield
and evaluates a sample dye using the following equation [59]:

2

Grad
Grad

η
η

=×

sample sample

ΦΦ

sample ref

2

ref ref

where Ф is quantum yield, grad is a slope of the curve total number of emitted pho-
tons to absorption at the wavelength of excitation, η is the refracting index, and ref
corresponds to reference dye and sample to the dye (nanoparticle) of interest.
In the relative method, both absorption and emission of the reference dye and the
sample are measured under identical conditions: the type of a cuvette (material, equal
optical path), excitation wavelength, slit size, integration time, detector, emission
range, and polarization have to be used for both the reference and the sample. for
emission, correction factors should be used if the reference and the sample emit in
significantly different spectral ranges (±50 nm). Caution has to be taken when NIR
dyes are measured using this method. Poor performance of standard detectors above
800 nm might lead to an erroneous conclusion that a dye with emission above 850 nm
has low quantum yield if the reference dye emits at shorter wavelengths. another
source of error lies in the difference of anisotropy between the reference and the
sample. To eliminate the potential discrepancy caused by the difference in anisot-
ropy, the polarizer at emission path should be set at the “magic angle” (54.7° from
the vertical). The detailed step-by-step procedure of measuring quantum yields is
given in several publications [60, 61].

6.7.2.3 Fluorescence Quantum Yield—Absolute Method Relative quantum
yield relies on the knowledge of Ф ref for a reference dye with similar absorption/
emission properties. If such a reference is not available, the QY must be measured by
absolute methods [62]. The most common current technique for measuring absolute
quantum yield utilizes an integrating sphere [63-66]. The main purpose of the sphere
is to minimize any special anisotropy of the emission and to eliminate refractive
index and polarization errors. The integrating spheres are often designed to fit in the
cuvette compartment of a fluorometer and are available for most of the conventional
fluorometers as an accessory [67]. In many high-end instruments, the integrating
sphere is external and connected to the instrument through a fiber-optic bundle
placed in the cuvette compartment (fig. 6.22). External design allows for larger
integrating spheres (typically 150 mm) with very homogeneous light flux and
minimal hot spots to achieve higher accuracy. Modern integrating spheres have
several ports for measuring quantum yields of samples in both liquid and solid form
[64, 68].
With the commercial availability of the integrating spheres, the absolute
method has increasingly been used to measure quantum yield of a variety of