Nanoscale chemical characterization of secondary protein structure of F-Actin using mid-infrared photoinduced force microscopy (PiF-IR)

The recently developed photoinduced force microscopy for mid-infrared (PiF-IR) offers high spectral resolution in combination with surface sensitivity and a spatial resolution in the range of a few nanometers. Although PiF-IR has primarily been applied to polymer materials, this technology presents significant potential for the chemical characterization of cellular structures approaching single-molecule sensitivity. We applied PiF-IR to differently polymerized F-Actin samples finding general agreement with FTIR spectra from the same samples. Single PiF-IR spectra of F-Actin show variations in the amide I band spectral region, which is related to secondary protein structure. Local variations are also seen in PiF-IR hyperspectra in this region. Such high sensitivity is a necessary requirement for discriminating Actin organization into bundles and other networks in cells and tissue. We applied PiF-IR to mouse liver tissue ex vivo. Single-frequency PiF-IR scans at three different IR frequencies show significant variations in local contrast. However, the presence of other proteins and the unique spatial resolution of PiF-IR pose a challenge to interpreting and validating such data. Careful design of model systems and further theoretical understanding of PiF-IR data far from bulk averages are needed to fully unfold the potential of PiF-IR for high-resolution chemical investigation in the Life Sciences.