Characterization of Biomaterials 241
surface characterization. They are convenient to use, low cost, yet capable of
obtaining preliminary information on the biocompatibility of biomaterials. The
most used thermodynamic method for biomaterial surface characterization is
wetting or contact angle experiments.
Contact angle analysis characterizes the wettability of a surface by measuring
the surface tension of a solvent droplet at its interface with a homogenous surface.
The phenomenon of contact angle can be explained as a balance between the force
with which the molecules of the liquid (in the drop) are being attracted to each
other (a cohesive force) and the atraction of the liquid molecues for the molecues
that make up the solid surface. An equilibrium is established between these forces,
the energy minimum. Contact angle is one of the most sensitive but inexpensive
surface analysis techniques and is capable of measuring 3–20
˚
A deep.
There are five common techniques that can be employed to measure the
contact angle: the Wilhelmy plate method, the sessile drop method, the capative
bubble method, the capillary rise method, and the tilted-drop measurement. The
details can be found in.
3,67,68
The technique chosen depends principally on the
geometry and location of the surface or coating to be studied. In all the methods,
the contact angle is the angle of the liquid at the interface relative to the plane
of the model surface. As application examples, contact angle analysis has been
successfully used to predict the performance of vascular grafts and the adhesion
of cells to surfaces.
10.3.5 Emerging Optical Methods for in vivo Analysis
Over the last two decades, great progress has been made on biomedical optical
imaging and spectroscopy technology development. Emerging novel optical
techniques enabled the imaging and analysis of biological tissues at micrometer
spatial resolution and up to millimeter depth below the tissue surface in spite of the
challenge of light scattering by biological tissues. These technologies are definitely
useful for monitoring implant biomaterials and MEMS devices in tissue or other
biological environment as well as for studying the interactions of biomaterials with
in vivo and other real biological environments. The most powerful biomedical
optical analysis technologies include confocal laser scanning microscopy (CLSM),
Raman spectroscopy and imaging, multi-photon excitation (MPE) microscopy, and
optical coherence tomography (OCT). CLSM and Raman techniques have been
introduced in previous sections, MPE microscopy and OCT will be discussed
below.
10.3.5.1 Multi-Photon Excitation Microscopy
Multi-photon excitation microscopy is a powerful tool that combines multi-
photon excitation fluorescence and confocal scanning microscopy to create high-
resolution, 3-D images of microscopic samples.
69
In traditional fluorescence mode
confocal microscopy, a single photon of light is used to excite a molecule from its
ground state to an upper excited electronic state to induce fluorescence emission
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242 H. Zeng
at longer wavelengths that are then used for image formation. Although the inter-
action probability is greatest for single-photon absorption, if two or more lower
energy (longer wavelength) photons arrive simultaneously, there is probability
that they can excite the molecule as long as the sum of the multi-photon energies
equals to the corresponding single-photon excitation related energy difference.
The multi-photon absorption probability is non-linear and increases with the
square of light intensity (I
2
) for two-photon absorption and as the cube for three-
photon absorption. For this reason, ultrafast lasers (hundreds of fs light pulses,
often Ti:Sapphire laser) are used to generate instantaneously super high power
density at a tightly focused spot in space to facilitate high probability multi-photon
absorption for multi-photon excitation microscopy applications.
Multi-photon absorption brings advantages over single-photon absorption.
With single-photon absorption, when a laser is focused to a point within a sample,
the sample may, because of the linear dependence on the light intensity for single-
photon absorption, fluoresce throughout the entire beam path. Using multi-
photon absorption, induced fluorescence occurs only at, or near, the focal point of
the beam thanks to the non-linear (quadric, cubic, or higher order) dependence on
the light intensity. Since the position of the focal point can be precisely determined,
multiphoton fluorescence can yield a great deal of information about specific
points below the sample surface. Furthermore, longer wavelengths, particularly
the near-IR, penetrate deeper in biological materials and are not scattered as
much as shorter wavelengths. These lead to a couple of advantages for MPE
microscopy over conventional fluorescence confocal microscopy: higher axial reso-
lution, greater sample penetration, reduced fluorescence photobleaching, reduced
photodamage and increased tissue and cell viability.
A conventinal CLSM can be modified to perform MPE microscopy with
two major changes: (1) the light source is changed to a ultrafast laser (usually
Ti:Sapphire) with very high peak power but low average power; (2) The confocal
detection aperture is removed because all of the fluorescent light originates from
Photodetector
CLSM
MPE
Pinhole
Dichroic
Mirror
Sample
Objective
Figure 10.10. Comparison of a conventional confocal microscope (CLSM) with a multi-
photon excitation (MPE) microscope. For color reference, see page 277.
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