Biomedical Engineering Reference
In-Depth Information
Confocal fluorescence microscopy is one of the most common ways to monitor the uptake and
localization of fluorescently labeled NMs. However, not all NMs can be easily fluorescently labeled.
Furthermore, the label may be released from the NMs into cells and, thus, the distribution of fluo-
rescence within the cells does not necessarily represent the presence or subcellular distribution of
the NMs. It is also possible that the label may change the biological behaviors of NMs.
TEM is frequently used to investigate the uptake, aggregation/agglomeration, and location of
NMs in cells [4] as a function of the size, shape, surface modification, or surface charge. For instance,
Gratton et al. [47] reported the internalization of monodisperse hydrogel particles into HeLa cells as a
function of size, shape, and surface charge. It was found that HeLa cells can internalize nonspherical
particles with dimensions as large as 3 μm by using different mechanisms of endocytosis.
By TEM, Belade et al. [48] showed TiO
2
and carbon black NPs were widely and rapidly accumulated
in 16HBE bronchial epithelial cells and MRC5 fibroblasts. Moreover, the NMs accumulated chiefly
as aggregates in cytosolic vesicles and were absent from the mitochondria or nuclei. Despite similar
accumulation patterns, TiO
2
aggregates had a higher size than carbon black aggregates. Interestingly,
they found that the intracellular NM accumulation was dissociated from the observed cytotoxicity.
This is common; the uptake of NMs into cells does not always cause toxic effects. In spite of the high
spatial resolution of conventional electron microscopy, the sample preparation for electron microscopy
studies is very complicated, including fixation, drying, sectioning, and staining [4,49].
An alternative option is cryo-TEM and cryo-SEM, where rapid freezing of the sample allows for
the analysis in a state similar to the hydrated state in the original solution [16,50]. The biological
sample is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid
freezing, usually plunge freezing in liquid ethane near the temperature of liquid nitrogen [51]. By
maintaining specimens at the liquid nitrogen temperature or colder, they can be introduced into the
high vacuum for measurements at variable temperatures. Thus, plunge freezing, cryo-transfer, and
imaging have the potential to produce a representative view of NMs in biological matrices. Cryo-
SEM can be applied to investigate the fine structures of bulk samples. Win et al. [52] studied the
effects of particle sizes and surface modifications on cellular uptake by cryo-SEM. The acquired
image clearly indicates that some NPs were found throughout the endoplasm and around the nucleus
of Caco-2 cells, and some were adsorbed on the cell membrane. In spite of the requirement of a
number of sample preparation steps and time-consuming measurements, the cryo-SEM and cryo-
TEM paves the way for a high-resolution image of aqueous specimens.
8.2.5 c
haracterIzatIoN
of
NM
s
IN
B
IologIcal
M
atrIces
By
r
aMaN
s
pectroscopy
Raman spectroscopy is well established for chemically identifying materials by sensing the vibra-
tion of chemical bonds, rather than performing elemental analyses. Raman spectroscopy is a laser-
based optical, label-free technique that excites vibrations of molecular bonds in a material. It relies
on the inelastic scattering, or Raman scattering, of a laser. The laser light interacts with molecular
vibrations, phonons, or other excitations in the system, resulting in the energy of the laser photons
being shifted up or down. The shift in energy gives information about the vibrational modes in the
system. Raman spectrum serves as a
molecular fingerprint
of a material, yielding information on
molecular bonds, conformations, and intermolecular interactions.
The Raman spectroscopy technique has been employed for biomedicine applications [53]. Each
biomolecule has a unique “fingerprint” of Raman peaks at well-defined frequencies, as they contain
a variety of molecular bonds (e.g., C-H, C=C, O-H, aromatic ring). Frequency shifts are recorded in
wavenumbers (cm
−1
), and biological molecules have vibrations in the range of about 600-3000 cm
−1
.
Thus, probing the change in the compositional and structural characteristics of a biological system
by the Raman spectroscopy technique can provide information on the state of the biological system
under investigation. Through enhancements with noble metal NPs, such as Au and Ag, surface-
enhanced Raman scattering (SERS) [54] has been applied to the detection of biomolecules such as
DNA [55], DNA/RNA mononucleotides [56], and proteins [57], to the labeling of cells [58,59] and
