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Observing single protein with infrared nanospectroscopy – Milestone toward ultra-high sensitivity and super-resolution infrared imaging

Figure 1: (Left) Scheme of near-field infrared spectroscopy measuring a single protein. (Right) The structure of the protein complex F1-ATPase and the subunit measured in this study. @ Institute for Molecular Science

Infrared vibrational spectra, often referred to as “molecular fingerprints”, are widely used for the structural and chemical analysis of various materials. The rapid development of nanotechnology in recent years has led to increasing demand for ultra-high sensitivity and super-resolution infrared imaging. However, conventional infrared spectroscopy is limited in terms of its sensitivity and spatial resolution. For example, even the best infrared microscope requires over a million proteins within tens of micrometer volume for obtaining an infrared vibrational spectrum, rendering it impossible to measure just a single protein.

An interdisciplinary research team, led by Jun Nishida (Assistant Prof.) and Takashi Kumagai (Associate Prof.) at Institute for Molecular Science, has successfully observed infrared vibrational spectra of single proteins using infrared near-field optical microscopy (Note 1). This method utilizes light confined at the nanometer scale, allowing for the detailed analysis of extremely small samples, which was challenging with conventional infrared spectroscopy.

In their study, the research team isolated a single protein, a sub-unit comprising a protein complex called F1-ATPase, on a gold substrate and performed near-field infrared spectroscopy measurements in an ambient environment. They successfully acquired the infrared vibrational spectrum of a single protein, representing a major advance that may lead to characterizing local structural organizations of individual proteins. Such information is particularly important for understanding the sophisticated functions of protein complexes and membrane proteins, offering deeper insights into their mechanisms and interactions. Furthermore, they have developed a new theoretical framework describing the nanoscale interactions between the infrared near field and protein. Based on the theory, the team was able to quantitatively reproduce the experimental vibrational spectra that they observed. These results will be invaluable for the chemical analysis of biomolecules as well as various nanomaterials, paving the way for a range of applications of nanoscale infrared spectroscopy. Details

Infrared near-field optical microscopy (known as nano-FTIR) has emerged and rapidly established itself in recent years as an ultra-sensitive and super-resolution imaging technique in the infrared regime. Nano-FTIR enables the characterization of material composition and structure at the nanometer scale—a capability that is significant in both fundamental scientific research and various industrial applications. Nano-FTIR has found widespread applications in different branches of science, and its technological progress would impact a variety of research fields, from biological science to material engineering, due to its capability to provide unique insights into material properties at the nanoscale.

The team in Institute for Molecular Science has developed a highly sensitive nano-FTIR system and has undertaken the challenge of measuring the infrared vibrational spectra of a single protein consisting only of 500 amino acid residues—a feat previously unachieved. Figure 1 shows a schematic diagram of the nano-FTIR system developed for this study. As a mid-infrared light source, the team employs a pulsed laser source with high stability and optimal bandwidth for vibrational spectroscopy applications. When the apex of a metallic tip in an atomic force microscope (AFM) is illuminated with the infrared pulse, a highly localized field called near field is formed at the nanoscale. The interaction between this near field and a protein deposited on a gold surface results in infrared scattering, which is detected by interfering with another pulse as in Fourier transform infrared spectrometer (FTIR) to yield a nanoscale infrared spectrum.

The scattering amplitude sensitively depends on the distance between the tip and the sample. The team took advantage of this unique property by modulating the tip‒sample distance at a frequency ωAFM and detecting the signal component that is modulated at the higher harmonics of the frequency (nωAFM) with a lock-in amplifier. This not only allowed the researchers to selectively detect the near-field scattering, but also to have access to highly localized near-field interactions confined to a single-nanometer scale by analyzing the higher harmonics (n) component.

Figure 2: Schematic diagram of the nano-FTIR system developed for this study. Yb oscillator: Ytterbium femtosecond laser, OPO: Optical Parametric Oscillator, DFG: Difference Frequency Generator, BS: Beam Splitter, OAP: Off-Axis Parabolic Mirror, HgCdTe: Infrared Detector, HPF: High Pass Filter, amp.: Preamplifier, Lock-in amp.: Lock-in Amplifier, sig.: Detected Signal, ref.: Reference Signal, ωAFM: Resonant Frequency of the Cantilever. @ Institute for Molecular Science

Figure 3(a) and (b) show the AFM image and infrared spectrum of a single protein, respectively. In the AFM image a profile reflecting a topography of a single protein is observed, and in the infrared spectrum a vibrational resonance originating from amide groups in the protein is detected at 1665 cm^-1. The amide vibrations are known to reflect secondary structures of proteins and has been extensively studied based on conventional FTIR spectroscopy and more advanced nonlinear infrared spectroscopy methods. In this work, the team demonstrated that the equivalent information can be acquired at the single protein level.

Figure 3: AFM image (a) and infrared spectrum (b) of a single protein. @ Institute for Molecular Science

The research team also demonstrated that the localization of the near-field scattering signal is significantly enhanced by analyzing the higher-order demodulation harmonics using the lock-in amplifier. Figure 4(a) shows a plot of the infrared near-field scattering amplitude against the distance between the tip and the sample in the nano-FTIR. The plot clearly shows that as the order of demodulation harmonics increases, the decay length of the infrared near-field scattering amplitude becomes shorter, indicating a more localized interaction. Additionally, as shown in Figure 4(b), the vibrational resonance in the infrared spectrum becomes stronger at the higher orders. These results demonstrate that higher-order demodulation probes more spatially localized near-field interactions. Figure 4(c) shows a graph plotting the decay length of the infrared near-field scattering amplitude against the order of demodulation, revealing that for n ≥ 6, the infrared near-field signal is localized to less than 5 nm.

Figure 4: (a) A graph plotting the infrared near-field scattering intensity (vertical axis) against the distance between the tip and the sample (horizontal axis). The graph shows demodulation orders from 2nd to 7th. (b) Infrared spectra measured at demodulation orders from 2nd to 7th. (c) A graph plotting the dependency of the decay length (vertical axis), obtained from the analysis of the graph in (a), against the demodulation order (horizontal axis). @ Institute for Molecular Science

The research team also performed theoretical analysis to rationalize the experimental results, focusing on the interaction between the infrared near field and single protein. The infrared near-field spectrum is obtained by calculating the polarization induced when an electrostatic field is applied to the system. Figure 5(a) depicts the different analytical models that were used for simulations. Figure 5(b) compares the theoretical infrared vibrational to the experimental results. The theory successfully reproduces the enhanced vibrational resonance at the higher demodulation harmonics. The further detailed comparison reveals that the model which closely replicates the actual situation (referred to as EMPD in the study) showed the best agreement with the experimental data.

Figure 5: (a) Analytical models used to reproduce the experimental results. DPD: Double Point Dipole Model, SMPD: Spherical Mirror and Point Dipole Model, EMPD: Elliptical Mirror and Point Dipole Model. (b) Calculated infrared spectrum of a single protein. (c) A graph plotting the peak values of the infrared spectrum (vertical axis) against the demodulation order (horizontal axis). Black diamonds: experimental data, black circles: DPD model, red circles: SMPD model, blue circles: EMPD model. @ Institute for Molecular Science

Significance of the Research Findings

Infrared spectroscopy is a versatile method used in physical, chemical, biological, and medical sciences for investigating structures, properties, and functions of materials. With the advancement of nanotechnology, nanoscale spectroscopy techniques have become increasingly important. This research represents a significant step towards the next generation of ultra-sensitive and high-resolution imaging using mid-infrared infrared light, which holds promise for applications in a variety of nanomaterials. Reference Sub-Tip-Radius Near-Field Interactions in Nano-FTIR Vibrational Spectroscopy on Single Proteins

Jun Nishida, Akihiro Otomo, Takanori Koitaya, Akitoshi Shiotari, Taketoshi Minato, Ryota Iino, and Takashi Kumagai


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