Biomaterials for MEMS

Characterization of Biomaterials 237

when the frequency of the IR light is varied (by monochromator), the IR light

after passing the sample is guided through an interferometer. The measured

signal is the interferogram. Performing a mathematical Fourier transform on

this signal results in a spectrum identical to that from conventional (dispersive)

infrared spectroscopy. FTIR spectrometers are cheaper than conventional spec-

trometers because building of interferometers is easier than the fabrication of a

monochromator in the IR wavelength range. In addition, measurement of a single

spectrum is faster for the FTIR technique because the information at all frequencies

is collected simultaneously. Because of its various advantages, virtually all modern

IR spectrometers are FTIR instruments.

A special technique called attenuated total reflection Fourier transform in-

frared spectroscopy (ATR-FTIR) is usually employed to characterize biomaterial

surfaces without using any distructive sample preparation procedures. ATR-FTIR

couples IR spectroscopy analysis with the physical phenomena of total internal

reflection to restrict the analyzed volume to within a few micronmeter depth of

the sample surface. For this technique, the incident electromagnetic waves (IR

light) are entirely reflected back into the initial medium. The electromagnetic

field is established in the second medium as an evanescent wave.

47

In attenuated

total reflectance (ATR) sampling mode the second medium is the material to be

studied and has higher refractive index than the initial medium. Information about

the molecular structure of the biomaterial, inter- and intra-molecular interactions,

crystallinity, conformation (e.g. proteins) and orientation of molecules can be

obtained through analysis of the detected infrared spectra.

19,48

It has also been

used to study the relationship of surface characteristics to cellular attachement.

49

Figure 10.8 shows an example of IR spectroscopy applications in characterizing

SU-8 surface modifications, aimed to functionize SU-8 for immobilization of

biomolecules on a Bio-MEMS structure.

50

10.3.3 Microspectroscopy and Spectral Imaging Methods

There is a clear need to understand not only what chemical and biological species

are present in a biomaterial but also how those species are spatially distributed. In

principle, if we perform any spectroscopy analysis discussed in Section 10.3.2 in

a point by point fashion over an area of the sample surface we will get a spectral

image, in which a spectrum curve is available for each pixel. This huge amount

of data set is often referred as a spectral cube. Analysis of the spectral cube can

generate 2-D distributions/images of the chemical constituents and structures of

the sample surface. In fact, XPS imaging and mass spectrometry imaging (SIMS

imaging and MALDI — matrix-assisted laser desorption ionization imaging) has

recently been realized in biomaterial researches.

51,52

However, widespread pratical

applications of spectral imaging techniques are often hamppered by the length

data acquisition time of point by point spectral mapping and the achievable spatial

resolution of the spectral measurments. Therefore, success of a spectral imaging

mode often depends or is pending on technical advancements to improve the

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238 H. Zeng

Figure 10.8. ATR-FTIR of SU-8 surface (a) before and (b) after surface modification

showing additional R–NH

2

group at 1607 cm

−1

. The other peaks are 960–1275 cm

−1

:

phenyl in plane bending; 1500–1600 cm

−1

: phenyl nucleus; 1660–2000 cm

−1

: aromatic

overtone of dCH; 2350 cm

−1

:CO

2

; 2750 cm

−1

: chelation intermolecular bond with C=O;

2850 cm

−1

:−CH

2

−; 2960 cm

−1

:−CH

3

−; 2930–3650 cm

−1

: water. With permission from.

50

detection sensitivity and shorten the data acquisition time. In the optics field,

advances on light sources/lasers, spectrographs, detectors, and fast computer data

processing have enabled the successful applications of IR spectral imaging and

Raman spectral imaging in biomaterials reseach. These two techiques will be

discussed in more details below.

10.3.3.1 Infrared Microspetroscopic Imaging

Infrared microspectroscopy measurement was first realized in the 1940s by cou-

pling an FTIR spectrometer to a light microscope.

53

Thus, it is also called FTIR

microspectroscopy. In early instruments, IR spectral imaging is acquired through

point-by-point mapping utilizing mechanical scanning of the sample. These

early instruments has been used for bone characterization and widely applied

to polymer research.

53

Infrared microspectroscopy has also been used to study

tissues and individual cells.

54,55

as well as to identify pathologic inclusions and soft

tissue calcifications.

56

Data collection using point-by-point mapping is relatively

slow and tedious. A technical improvement, the coupling of the FTIR microscope

with an array detector for FIIR imaging changed the way data was collected and

processed.

57,58

The entire field of view of the sample is analyzed simultaneously

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