Biomaterials for MEMS

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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