Abstract:
Fourier-transform infrared (FTIR) spectroscopic imaging is a widely used method for studying the chemistry of proteins, lipids, and DNA in biological systems without the need for additional tagging or labeling. This technique can be especially powerful for spatially resolved, temporal studies of dynamic changes such as in vivo protein folding in cell culture models. However, FTIR imaging experiments have typically been limited to dry samples as a result of the significant spectral overlap between water and the protein Amide I band centered at 1650 cm-1. Here, we demonstrate a method to rapidly obtain high quality FTIR spectral images at submicron pixel resolution in vivo over a duration of 18 h and longer through the development and use of a custom-built, demountable, microfluidic-incubator and a FTIR microscope coupled to a focal plane array (FPA) detector and a synchrotron light source. The combined system maximizes ease of use by allowing a user to perform standard cell culture techniques and experimental manipulation outside of the microfluidic-incubator, where assembly can be done just before the start of experimentation. The microfluidic-incubator provides an optimal path length of 6-8 μm and a submillimeter working distance in order to obtain FTIR images with 0.54-0.77 μm pixel resolution. In addition, we demonstrate a novel method for the correction of spectral distortions caused by varying concentrations of water over a subconfluent field of cells. Lastly, we use the microfluidic-incubator and time-lapsed FTIR imaging to determine the misfolding pathway of mutant copper-zinc superoxide dismutase (SOD1), the protein known to be a cause of familial amyotrophic lateral sclerosis (FALS).
PMID: 25965274 [PubMed – as supplied by publisher]