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.

Automated summary

The recently developed PiF-IR microscopy offers high spectral resolution, surface sensitivity, and nanoscale spatial resolution. It has the potential to characterize cellular structures at the single-molecule level. PiF-IR was applied to polymerized F-Actin samples and mouse liver tissue, showing variations in contrast. However, interpreting and validating the data is challenging due to the presence of other proteins and the unique spatial resolution of PiF-IR.