Characterization of Protein Structural Changes in Living Cells Using Time-Lapsed FTIR Imaging

Area of Science:

• Biophysical Chemistry
• Cellular Biology
• Spectroscopy

Background:

• Fourier-transform infrared (FTIR) spectroscopic imaging analyzes biomolecules like proteins, lipids, and DNA without labeling.
• Current FTIR imaging is limited to dry samples due to water's spectral interference with protein Amide I bands.
• In vivo studies of dynamic cellular processes like protein folding are hindered by these limitations.

Purpose of the Study:

• To develop a novel method for high-quality, in vivo FTIR spectroscopic imaging of biological samples over extended periods.
• To overcome the spectral interference of water in FTIR imaging of live cells.
• To enable detailed spatiotemporal analysis of protein misfolding pathways in cellular models.

Main Methods:

• Development of a custom-built, demountable microfluidic-incubator for FTIR microscopy.
• Coupling the incubator with a focal plane array (FPA) detector and synchrotron light source for high-resolution imaging.
• Implementation of a novel water spectral distortion correction method for cellular samples.
• Time-lapsed FTIR imaging of mutant copper-zinc superoxide dismutase (SOD1) in a cell culture model.

Main Results:

• Achieved submicron pixel resolution (0.54–0.77 μm) in vivo FTIR imaging over 18+ hours.
• Successfully minimized spectral overlap between water and protein Amide I bands.
• Demonstrated effective correction for water concentration variations across cell cultures.
• Visualized the misfolding pathway of mutant SOD1, a protein implicated in familial amyotrophic lateral sclerosis (FALS).

Conclusions:

• The developed microfluidic-incubator system significantly advances in vivo FTIR spectroscopic imaging capabilities.
• This technique allows for unprecedented temporal and spatial resolution of cellular chemistry and dynamics.
• The method provides a powerful tool for investigating disease mechanisms, such as protein misfolding in neurodegenerative diseases.