Characterization Techniques of Living Cells Encapsulated with Nanomaterials - Cell Surface Engineering: Fabrication of Functional Nanoshells - page 81

particular, a molecular spectroscopic technique can quickly provide very
valuable information about the molecular environment of the material
placed on or in the cell. In this chapter, the characterization techniques used
in living-cell modifications are discussed. The techniques are grouped into
three categories as imaging, spectroscopic, and other techniques and sum-
marized by giving examples from the literature.

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5.2 Imaging Techniques
Since the aim is to alter cell surface functionality, the foreign materials, such
as nanoparticles and polymers, are targeted to be placed onto the cell sur-
face. Therefore, the imaging is performed to visualize the location of the
materials placed on the surface of a cell. There are two important parameters
for decision making for which imaging technique should be used for char-
acterization. These parameters are the resolution limit of the technique and
the nature of the materials such as a polymer or a nanomaterial attached to
the cell surface. In a microscopic technique, a light beam, electron beam or
physical probe is used for imaging. When light is used as the probe, it is
called optical microscopy, when an electron beam is used, it is called elec-
tron microscopy (EM) and when a tip or tapered fiber optic is used, it is
called scanning-probe microscopy (SPM).
A light microscope can be used to gather spectroscopic information. For
example, fluorescence or Raman scattering are commonly collected using an
optical microscope. In conventional light microscopy, all scattered light
originating, regardless whether from the focal plane and out-of-focus plane,
is collected. However, it is possible to collect scattered light for imaging from
the focal plane. In confocal microscopy, the image is collected from the focal
plane. Only light collected from the focal plane is used in imaging and out-
of-plane light is removed. Therefore, most of the collected image originates
from the focal plane in confocal microscopy. Since light is used as a probe,
the spatial resolution is mostly defined by the diffraction of the light. The
spatial resolution can given with a formula of S ¼ 0.61 l/NA, where l is the
wavelength of the light used for imaging and NA is the numerical aperture of
the objective used. Although this formula gives the theoretical limits of how
a light beam can be focused on an area, the diameter of the light beam
impinging onto the sample from the objective could be greater than 1 mm.
Therefore, it is important to realize the size of the material placed onto the
cell surface. If it is a nanometer-sized material without fluorescing or plas-
monic property, it cannot be visualized with an optical microscope.
Fluorescence spectroscopy is a molecular spectroscopic technique based
on the excitation of a molecule at a certain wavelength and recording the
emitted light with a detector. Not every molecule has a fluorescence property
and it is strongly related to the chemical structure of a molecule varying from
molecule to molecule. The florescence property of molecules is utilized in
fluorescence and confocal microscopies. Fluorescence microscopy can sim-
ply be defined as optical microscopy with a proper light source for excitation

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