Hyperspectral interferometry: Sizing microscale surface features in the pine bark beetle

A new method of interferometry employing a Fabry-Perot etalon model was used to locate and size microscale features on the surface of the pine bark beetle. Oscillations in the reflected light spectrum, caused by self-interference of light reflecting from surfaces of foreleg setae and spores on the elytrum, were recorded using white light hyperspectral microscopy. By making the assumption that pairs of reflecting surfaces produce an etalon effect, the distance between surfaces could be determined from the oscillation frequency. Low frequencies of less than 0.08 nm(-1) were observed in the spectrum below 700 nm while higher frequencies generally occupied wavelengths from 600 to 850 nm. In many cases, two frequencies appeared separately or in combination across the spectrum. The etalon model gave a mean spore size of 3.04 ± 1.27 μm and a seta diameter of 5.44 ± 2.88 μm. The tapering near the setae tip was detected as a lowering of frequency. Spatial fringes were observed together with spectral oscillations from surfaces on the exoskeleton at higher magnification. These signals were consistent with embedded multi-layer reflecting surfaces. Possible applications for hyperspectral interferometry include medical imaging, detection of spore loads in insects and other fungal carriers, wafer surface and subsurface inspection, nanoscale materials, biological surface analysis, and spectroscopy calibration. This is, to our knowledge, the first report of oscillations directly observed by microscopy in the reflected light spectra from Coleoptera, and the first demonstration of broadband hyperspectral interferometry using microscopy that does not employ an internal interferometer.