Nanoparticles for Bioimaging: Analytical Characterization and Measurements - Nanotechnology for Biomedical Imaging and Diagnostics - page 170

advantage of the integrating sphere is especially apparent when there is a need to
measure absorption spectra of nanoparticles in very turbid media such as a cell
suspension or in blood. a variation of the method uses an integrating cavity
absorption meter [41] shown in figure 6.17. Since the light path in the cavity is sig-
nificantly longer due to multiple reflections and reabsorption events, molar absorp-
tivities are corrected as described by Hodgkinson et al . [42] and fry et al . [43] with
the latter implemented in some modular spectrophotometers (such as Clarity from
olis, Inc., United States).
6.7.1.2 Fluorescence Spectrophotometers low sensitivity in NIR is not critical
for absorption spectrophotometers, allowing absorption measurement in NIR with
acceptable accuracy (many conventional spectrophotometers measure up to 1100 nm).
However, several considerations have to be addressed before a conventional fluores-
cence spectrophotometer can be modified to detect NIR emission. Basically, all of
the wavelength-dependent components of the instrument have to be evaluated for
performance in the NIR spectral range. These components consist of the excitation
source, gratings in the excitation and emission spectrometers, detector, polarizers,
and fiber optics. Due to various graded NIR parts and modules, such systems are
often customized and generally associated with higher cost. an example of an instru-
ment for nanoparticle research (Nanolog, Horiba, Inc.) is given in figure 6.19 with
some of the modules described in the following text.
6.7.1.3 Excitation The excitation source in a typical fluorescence spectropho-
tometer is a Xe arc lamp. This lamp has a substantial number of sharp peaks between
700 and 1000 nm (fig. 6.20) that could introduce abnormalities into the spectra.
Hence, it requires parallel recording of the light input using a photodiode and
subsequent correction of the emission spectra. alternatively, a tungsten lamp can be
utilized as an excitation source because of its broad and smooth output in the NIR
range (up to 2000 nm).
6.7.1.4 Gratings Conventional excitation gratings on fluorescence systems are
usually diffraction-type gratings that are optimized (blazed) in the UV range (typi-
cally 300 nm), while the emission gratings are blazed in the visible range (typically
500 nm). for NIR performance, both gratings should be replaced with blazed grat-
ings at longer wavelengths. a NIR configuration includes the excitation grating
blazed at greater than equal to 500 nm and the emission grating blazed in red or NIR
(500-1000 nm). Some advanced models are equipped with kinematically inter-
changeable gratings for higher flexibility for allowing the conversion of a standard
system into a NIR-dedicated unit manually or via software interfaces.
6.7.1.5 Detectors Most research emission spectrophotometers are currently
equipped with a R928 or R928P (for photon counting) Si-type photomultiplier tube
(PMT). Introduced in 1974 by Hamamatsu, Inc. (Japan), this PMT with relatively
high sensitivity to red and NIR ranges made an “epoch-making event” [44] in
optical spectroscopy. However, the performance of R928(P) rapidly drops above