Page 329 - Vibrational Spectroscopic Imaging for Biomedical Applications

Raman Imaging of Str ess Patterns in Biomaterials 303

Stress assessments in the Raman spectroscope are possible because
of the PS effect. 12,19,20 The PS effect may be simply defined as the shift in
the wavelength of a spectroscopic transition in a solid in response to an
applied strain or stress. This definition has been given as a general one,
independent of the spectral signal arising from luminescence or Raman
scattering. In linear elastic materials, the PS effect can be represented
with a linear (tensorial) relationship between the second-rank stress
tensor and the observed Raman wavenumber shift with respect to an
unstressed (reference) wavenumber value characteristic of a selected
Raman band. Upon applying preliminary calibrations, it is possible to
determine the proportionality constant (i.e., a constant usually referred
to as the PS coefficient) between various kinds of stress tensors (e.g., uni-
axial, equi-biaxial, etc.) and wavenumber shifts of each selected Raman
band. Based on this knowledge, it is then possible to quantitatively ana-
lyze unknown stress states. A rigorous treatment of the tensorial PS for-
malism applied to Raman bands, a description of the related calibration
methods and a verification of its validity in various natural and synthetic
biomaterials have been given elsewhere. 21
Throughout our studies, Raman spectra were collected with a triple
monochromator spectrometer (T-64000, ISA Jovin-Ivon/Horiba Group,
Tokyo, Japan) equipped with a charge-coupled detector (high-resolution
CCD camera). All the spectra were recorded at room temperature. The
laser excitation source was a monochromatic blue line emitted by an Ar
laser at a wavelength 488 nm or by a Kr laser at a wavelength of 614 nm.
The optical microscope was connected to a video monitor that allowed
scanning of the sample surface to locate the selected location for the
spectroscopic measurement. The wavenumbers of Raman band-
maxima were obtained by fitting the CCD raw data to mixed gaussian/
lorentzian curves with commercially available software. Samples were
placed on a mapping device (lateral resolution of 1 μm), which was con-
nected to a personal computer to drive highly precise in-plane displace-
ments (along both x and y axes) on the sample. Given the curved nature
of the investigated surfaces, an autofocus device was adopted through-
out automatic mapping experiments. Confocal experiments were con-
ducted with focusing the waist of the laser beam either on the surface or
on subsurface planes of the investigated biomaterial through an optical
lens (100×). Then, the Raman signal was re-focused onto a small confo-
cal (pinhole) aperture that acted as a spatial filter (pinhole aperture-
diameter of 100 μm), passing the signal excited at the beam waist, but
substantially eliminating fluorescence signals produced at other points
above and below the beam waist. The filtered signal then returned to
the spectrometer (via the probe head) where it was dispersed onto the
CCD camera to produce a spectrum. In practice, a pinhole aperture was
placed in the optical train of the spectrometer and used to regulate the
rejection of out-of-focus light. The penetration depth of the laser
depended on the transparency of the investigated material as well as
on the selected pinhole aperture. Confocal microscopy was used to