Page 74 - Vibrational Spectroscopic Imaging for Biomedical Applications

50 Cha pte r T w o

minced into small pieces. When these cartilage pieces are incubated
with ATP, they form CPPD crystals. When they are incubated with
45
β-glycerophosphate (βGP), they form BCP crystals. A similar model
involves the use of chondrocytes in high-density monolayer cultures.
When these cell layers are exposed to ATP, CPPD crystals are gener-
45
ated, and with βGP exposure they generate BCP crystals. Small
membrane bound extracellular organelles known as articular carti-
lage matrix vesicles (ACVs) can be isolated from normal articular car-
tilage. ACVs also generate crystals in a similar manner to chondrocytes
and cartilage. 46

2.4.4 Synchrotron-Based FTIR Microspectroscopy Spectral
Analysis of Calcium-Containing Crystals
We wondered if the use of a synchrotron beam with FTIR spectral
microanalysis could increase the sensitivity of this modality so that
we could conclusively identify small sparse calcium-containing crys-
tals in a variety of settings. Drop-sized samples from in vitro models
or human synovial fluids were placed onto Kevley IR reflective slides
and examined with plain and compensated polarized light micros-
copy to locate birefringent or dense materials. These areas were pho-
tographed and marked so that the same areas could be examined
with synchrotron-based FTIR microspectroscopy. The samples were
measured with a Thermo Fisher Continuμm Fourier Transform-IR
(FT-IR) microscope coupled to the IR beamline at the Synchrotron
Radiation Center (SRC) in Stoughton, Wisconsin. Measurements were
taken in reflectance, acquiring reflection-absorbance results. Both
individual spot-measurements and spatially resolved maps of the
2
samples were measured with apertures ranging from 8 to 15 μm . The
number of scans was selected to optimize the SNR and visible images
were collected concurrently. The IR results were visually compared to
reference spectra of multiple forms of calcium-containing crystals.
As shown in Figs. 2.15 and 2.16, we could conclusively identify
CPPD and BCP crystals in human samples as well as in ACV and cell
47
culture models of crystal formation. The crosshairs in the visible
images indicate the points at which the measurements were obtained.
2
The top sample spectrum was collected with a 10 × 10 μm aperture
and 64 scans, while the bottom sample spectrum was collected with
2
an 8 × 8 μm aperture and 64 scans. For the latter crystal, the sample
2
is clearly smaller than the 8 × 8 μm aperture, but is completely within
the illuminated field of view. The IR signatures produced by the
synovial fluid crystals clearly match the standard spectra of BCP and
CPPD crystals, respectively. The additional peaks on the synovial
fluid crystal spectra suggest the presence of biologic material mixed
with the crystals.
Chondrocyte monolayers were incubated with ATP for 72 hours.
A crystal from these monolayers was compared with a M-CPPD