Infrared microscope detection of particulate inclusions in tissues

Small biomaterials wear particles frequently associated with implant failure and other "foreign body" inclusions are often noted to trigger adverse tissue responses, usually found within granulomatous masses in conventional biopsy microscopic 5microm thick sections. Differential identification of such fugitive particulate matter, limited by standard light microscopy of fixed, stained tissue sections, can be accomplished by InfraRed (IR) Microscopy if appropriate sample preparations are used. The purpose of this investigation is to employ visible digital imaging and aperture-limited IR spectroscopy at repeatable coordinates of thin film and tissue biopsy sections, in order to select optimal conditions for identification of reference and unknown particulate matter. A pilot study employed thin films of collagen and gelatin containing IR-identifiable reference prosthetic wear particles of polyethylene and polytetrafluoroethylene, as well as IR-inactive titanium as a scattering-only control, on IR-transparent germanium internal reflection prisms to record IR spectra in microscopic transmission (T), microscopic reflection (R), and multiple attenuated internal reflection (ATR) modes for all specimens. It was determined that square image areas of 20 micrometers on a side were routinely suitable for acquisition of excellent T- and R-IR spectra for distributed small particles not otherwise identifiable by ATR-IR. Finely distributed smaller particles within 15x15 micrometer apertures also produced excellent T- and R-IR spectra, but 10x10 micrometer apertures were too limiting in all cases. As an interpretive aid, an inventory and IR microscopy library of known particulate standards, alone and in gelatin surrogate tissue matrices, was produced and used to authoritatively identify and segregate five different IR microscopy-inspected materials---calcium carbonate, calcium phosphate, sodium phosphate, silica, and alumina---as confirmed by subsequent scanning electron microscopy and energy dispersive x-ray spectroscopy of the same preparations. IR microscopy also was able to separate clearly thin film residues from diesel versus biodiesel fuels on the basis of unique IR-active ester bands in the biological specimens.;De-identified human dental tissue biopsy specimens were prepared on glass, IR-reflective coated glass, and germanium prisms as paraffin-embedded sections of increasing thicknesses and examined by both visible light microscopy and IR spectroscopy for location and identification of particulate matter. It was determined that non-hydrocarbon fugitive particulate matter could be identified by IR Microscopy in 2-, 5-, and 10-micrometer thick paraffin sections, with the T-IR spectroscopic mode producing better spectra than the R-IR mode for specimens on germanium substrata. Even on coated glass specimens, however, R-IR was superior to conventional transmission IR or ATR in analysis of discrete particulate inclusions within tissue volumes and not in intimate contact with germanium prism surfaces. IR-reflective coated glass is a suitable substratum for routine R-IR microscopic analysis of biopsy thin sections.