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Algal Cells, Cartilage, and IRENI 55

tetracyclines are also fluorescent. Taken together, these findings led to
ask if tetracycline could be used as a fluorescent marker of synovial
fluid BCP crystals. Using synthetic BCP crystals, we showed that the
addition of tetracycline allowed us to visualize BCP-containing parti-
cles in synovial fluid. We used synchrotron FTIR spectral analysis to
prove the presence of BCP crystals in clinical synovial fluids, and then
showed that these fluids also contained fluorescent particles using
52
tetracycline staining. This novel assay for BCP crystals will require
further testing in the clinic, but is an exciting advance over our current
identification methods. This work could not have been performed
without the synchrotron FTIR analysis to prove that indeed we were
identifying BCP crystals.

2.5 Future Directions: In Vivo Kinetics of Pathological
Mineralization and Phytoplankton Adaptation
In the previous sections of this chapter, we have described the devel-
opments of (1) IRENI, a new synchrotron facility for rapid IR imaging
at the diffraction limit covering the mid-IR frequency range from
–1
4000 to 950 cm , (2) a new flow chamber to maintain biological spec-
imen in a hydrated environ that makes in vivo IR imaging feasible,
and (3) a biomedical application of synchrotron IR microspectroscopy—
studying calcium-containing crystals in cartilage from human sam-
ples and model systems. This combination of advances will allow
collection of high-quality IR hyperspectral cubes within 1 minute,
2
probing 40 × 60 μm per experiment with a spatial oversampling of at
least 2 to 1 for all wavelengths of interest. Future experiments will
bring these developments together to study the pathological mineral-
ization in cartilage by collecting time-resolved images of samples in
vivo. Using chemometrics we can quantify collagen by using the
amide I peak, denatured collagen and estimated proteoglycans, and
observe crystal formation in a single area of a specific specimen. We
propose to see if matrix changes precede or follow crystal formation
and whether we could quantify alterations in lipid, proteoglycan, or
denatured collagen. Other projects will include further studies of
phytoplankton that are fully hydrated and maintained in a controlled
medium, monitoring adaptation to different environmental stimuli.

Acknowledgments
This work was supported by the NSF under Award Nos. CHE-0832298
(CJH, MN, MG, SR), DMR-0619759(CJH, MN), NIH grant AR-R01-
056215 (AKR), and by the Research Growth Initiative (RGI) of the Uni-
versity of Wisconsin-Milwaukee (CJH, MG). Part of this work is based
upon research conducted at the SRC, University of Wisconsin-Madison,
which is supported by the NSF under Award No. DMR-0537588.