A research group led by Nodoka Wakita, a second-year master's student in the Program of Life and Materials Chemistry at Nagoya Institute of Technology, Assistant Professor Tatsuro Nishikino and Associate Professor Yuji Furutani of the Department of Life Science and Applied Chemistry at Nagoya Institute of Technology, and Professor Mitsumasa Koyanagi and Professor Akihisa Terakita of the Graduate School of Science at Osaka Metropolitan University has developed a technique for visualizing the distribution of protein structures by performing infrared imaging of living nematodes used as model organisms. The findings were published in ACS Omega.
Provided by Nagoya Institute of Technology
To date, fluorescence microscopy using fluorescent dyes or fluorescent proteins has been the primary method for observing proteins and lipids in living organisms. However, these approaches require molecules to be artificially labeled, which can potentially alter their native biological state.
Infrared imaging has attracted attention as a technique that can visualize proteins and lipids without staining by exploiting the intrinsic infrared absorption properties of biomolecules. However, its application to living organisms has been limited by two major challenges: the strong absorption of infrared light by water, which is abundant in biological tissues, and movement of the organisms during measurement.
To enable infrared imaging of live nematodes, the research team addressed both of these challenges. First, to reduce interference from water, the researchers used a buffer containing heavy water (D2O) instead of ordinary water (H2O). They also used sodium azide to temporarily immobilize the nematodes and established conditions under which locomotion and reproductive capacity recovered after imaging.
For infrared imaging, the team employed a Fourier-transform infrared (FT-IR) microscope equipped with a focal plane array (FPA) detector and successfully acquired infrared spectral images of individual nematodes within a few minutes. The resulting spectra were used to visualize the distribution of lipids and proteins, confirming, among other findings, that lipids are concentrated around the intestinal region. Furthermore, by applying second-derivative analysis to the protein-derived amide I band, the researchers separated spectral components associated with α-helical and β-sheet protein structures and visualized their respective spatial distributions.
The analysis revealed that the proportion of β-sheet structures is lower in the nematode head region. This is thought to reflect the abundance of muscle proteins such as myosin and actin in the pharyngeal muscles of the head, where α-helical structures are particularly prevalent.
A key feature of the newly developed technique is its ability to visualize not only the distribution of proteins and lipids within living organisms, but also differences in protein structure, all without the need for molecular labeling.
Structural abnormalities in proteins are closely associated with many neurodegenerative disorders, including Alzheimer's disease and prion diseases. In particular, amyloid aggregates formed by the accumulation of β-sheet structures are believed to play important roles in disease onset and progression.
This study presents a new label-free method for visualizing the spatial distribution of protein structures within living organisms and could contribute to future research on the mechanisms of neurodegenerative diseases as well as drug discovery. Because the technique uses infrared light, it is less likely to interfere with optical manipulation technologies that rely on visible light, such as optogenetics. The researchers therefore expect it could lead to a new bioimaging approach in which biological functions are controlled with light while molecular states are simultaneously observed using infrared spectroscopy.
Going forward, the team plans to apply the technique to nematode models expressing amyloid proteins associated with neurodegenerative disorders such as Alzheimer's disease, with the aim of observing protein aggregation processes in a label-free manner.
Journal Information
Publication: ACS Omega
Title: Minimally Invasive Infrared Imaging of Caenorhabditis elegans Enabled by Reversible Chemical Immobilization and Second-Derivative Spectral Mapping
DOI: 10.1021/acsomega.6c03485
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