Page 54 - Vibrational Spectroscopic Imaging for Biomedical Applications

Algal Cells, Cartilage, and IRENI 31

Another important issue with respect to working with biological
samples in vivo is to maintain living samples, especially for samples
that require water, such as phytoplankton. Many IR transparent
materials used for commercial, conventional flow cells are hygro-
scopic or poisonous, and thus not suitable for this application. Fur-
thermore, they are dependent on thick windows, which cannot be
used with the high-magnification objectives required for high-quality
spatially resolved images in both the IR and visible bandwidths. The
Section 2.2 of this chapter presents results using a newly designed
flow chamber that addresses these issues and maintains hydrated,
living cells in a 15-μm layer of water. 18
The third part of this chapter is based on a recent biomedical
application of synchrotron-based IR studies of minerals embedded
in cartilage tissues of osteoarthritic joints. The data allowed spectral
identification of small (~1 to 10 μm) calcium containing crystals
embedded in arthritic tissues and model systems, including calcium
pyrophosphate dihydrate (CPPD) and basic calcium phosphate
(BCP) crystals that are common components of osteoarthritic joints
and contribute to the irreversible tissue destruction seen in this form
of arthritis. 24
In the summary, future experiments will be described. These
experiments will combine all of the above advances, studying in vivo,
biologically important samples at diffraction-limited spatial resolu-
tion for all mid-IR wavelengths. Importantly, these results will be
2
achievable on a rapid timescale of 1 minute per 40 × 60 μm image
2
with 0.54 × 0.54 μm pixels, such that dynamic processes can be fol-
lowed with chemical specificity.

2.2 IR Environmental Imaging
IR Environmental Imaging (IRENI), a new IR beamline at the Synchro-
tron Radiation Center, Stoughton, Wisconsin, is designed to accept a
swath of radiation from the synchrotron and reshape the beam to illu-
2
minate a 40 × 60 μm sample area of an IR microscope. Traditionally,
synchrotron beamlines extract one beam that illuminates the sample
plane with a two-dimensional gaussian profile (FWHM of between
10 × 10 μm to 15 × 15 μm ). For the new beamline, 12 overlapped beams
2
2
illuminate a Bruker Hyperion 3000 microscope that is equipped with
an IR-sensitive FPA detector, where each pixel represents 0.54 × 0.54
2
μm area of the sample creating oversampled images at all wavenum-
bers in the mid-IR. This facility will provide the opportunity to obtain
chemical images with diffraction-limited resolution of the illuminated
area in under a minute.
Synchrotron radiation is inherently a broadband, bright, and sta-
ble source of IR radiation. 26,27 In the mid-1990s, the first experiments
28
using synchrotron radiation coupled to a commercial and a home-
built microscope 29,30 were reported. Since then, there has been a large