Biomaterials for MEMS

232 H. Zeng

The spatial resolution of CLSM is diffraction limited, i.e. at the order of the

wavelength of light used for imaging. Thus, micron and sub-micron resolutions

are readily achievable with CLSM.

31

The actual useful depth of CLSM is limited

to 100–200 μm, even with the most penetrating near IR illumination. Different

imaging modalities are available with CLSM depending on the types of optical

signals utilized for imaging formation. For example, reflectance confocal imaging

mode uses elastic backscattering light, while fluorescence confocal imaging utilizes

sample fluorescence emission. Confocal Raman imaging can be performed by

utilizing inelastic (Raman) scattering.

CLSM applications in biomaterials research is expanding with advantages

such as convenience of use, in that no special sample preparation is required as

compared to electron microscopy, and high spatial resolution than conventional

light microscopy. Confocal microscopy can resolve details at interfaces that

could previously only be seen using electron microscopy of specially processed

specimens.

32

To assess the biocompatibility of poly-β-hydroxy butyric acid (PHB)

polymer, CLSM has been used to study the structural organization of cellular

molecules involved in adhesion using osteoblastic and epithelial cell lines.

33

Fig-

ure 10.5 shows example confocal images from this study demonstrating that for

cells cultured on PHB polymer, the organization of the actin cytoskeleton was

impaired. To devise better strategies for developing biomaterials optimized for

particular therapies and treatments, a novel, CLSM based 3-D screening method

for analyzing interactions between cells and new biomaterials has been developed

to cut initial search times by more than half.

34

Recent technology advancement has

resulted in a reflectance mode CLSM that is capable of acquiring video rate cellular

resolution images from human skin in vivo.

35

This could presumably be adopted

to study the interactions of MEMS biomaterials with biological tissues in vivo in

animal models.

10.3.2 Spectroscopy Methods

Spectroscopy methods are routinely used to analyze the constituent elements and

chemical structures of biomaterial surfaces. In this section, we will discuss the

most commonly used three types of traditional spectroscopy techniques, namely,

X-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS),

and attenuated total reflection Fourier transform infrared spectroscopy (ATR-

FTIR).

10.3.2.1 X-ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) also known as ESCA (electron spec-

troscopy for chemical analysis) is the most extensively used technique for the

characterization of biomaterial surfaces,

1,3

It is used to determine the surface

chemistry of a biomaterial in its “as received” state or after surface modifica-

tions, e.g. adsorption and retention of chemicals such as antibiotics and bonding

agents,

15

for the detection of immobilized proteins,

36

understanding the chemistry

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Characterization of Biomaterials 233

Figure 10.5. Fibronectin formation (green) and actin filament formation (red) in epithelial

mHepR1 cells cultured on the biomaterial PHB (below) compared to control cultures

(above). Note that in control cultures the actin filaments are structured in fibrils showing

colocalization with fibronectin (arrow head) in contrast to PHB cultured cells, where the

organization of actin filaments is more irregular and the colocalization of fibronectin with

actin is lost (arrow head) (CLSM images taken with LSM 410, Carl Zeiss Jena). With

permission from.

33

For color reference, see page 276.

of the structure, formation and stability of plasma treated surfaces,

19

as well as for

the characterization of the steps of formation of thin coatings.

37

XPS spectra are obtained by irradiating a material with a beam of aluminium

or magnesium X-rays while simultaneously measuring the kinetic energy (KE) and

number of electrons that escape from the top 1 to 10 nm of the material being

analyzed.

38

XPS requires ultra-high vacuum (UHV) conditions. The kinetic energy

of the emitted photoelectrons is unique for different elements and is sensitive to the

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