UV and visible microscopy “see” electronic transitions inside highly absorbing molecules that contain aromatic rings or highly conjugated carbon-carbon double bonds and carbonyl groups. Ellen V. Miseo, PhD, scientific marketing manager at Analytical Answers (Woburn, MA), explains that, as with their nonmicroscopic counterparts, UV and visible microscopy confirm chemical characteristics but lack granularity. “UV and visible spectra are big lumps that work best when you know what you’re looking for.”
Infrared (IR) microscopy is based not on electronic transitions but on vibrational transitions between atoms in a molecule, with each bond having its own characteristic IR activity. “IR is specific for compounds, which is why you can ask ‘What is it?’”
Infrared microscopy was invented in the late 1950s but did not catch on until the 1970s, when the availability of Fourier transform IR (FT-IR) provided higher throughput and improved signal-to-noise ratios. IR microscopy analyzes down to about 12 microns and is sensitive for chemical detection down to concentrations of about 0.01 percent.
Jasco’s Richard Larson, PhD, has published extensively on novel tweaks to microscope-based imaging, particularly attenuated total reflectance (ATR) FTIR. ATR-FTIR works like conventional FT-IR but does not require extensive sample preparation. ATR exploits the complete reflection of the addressing IR beam through an ATR crystal—a highly IR-permeable material with a high refractive index that causes beams to reflect off surfaces and into the detector. ATR crystal materials include zinc selenide, germanium, and silicon. ATR improves FT-IR resolution down to about 0.4 microns. Automated stages and mapping software enable acquisition of chemical “maps” by sampling discrete points at specific wavelengths and reconstructing a spectral image based on peak location, height, and area.
ATR-FTIR microscopy serves industries interested in chemical characterization at very small scale, such as polymer laminates, critical part failures, toxicology, paints, and personal care products, and defect analysis in finished products.