S37 - Influence of Cell Fixation Timepoint on Colorectal Cancer Cells by Optical Photothermal Infrared Imaging

How cell fixation timepoint affects molecular information obtained by O-PTIR to optimize sample preparation

Abstract Text:

Biological systems often exhibit time dependent changes based on chemical environment, potentially altering expected results and assays. Accounting for factors that affect the chemical environment over time is important to understanding cellular response and function. Recently, optical photothermal infrared (O-PTIR) spectroscopy has emerged as a technique to image single cells with submicron resolution. O-PTIR spectroscopy is a technique that utilizes a pump-probe system where the absorbance of infrared light excites molecular vibrations, causing a photothermal response that is measured by the change in scattering of visible light off the sample. Because the probe beam is a single visible wavelength, the spatial resolution is decoupled from the infrared beam. By tuning the infrared frequency, hyperspectral images can be collected with improved spatial resolution. This is advantageous for studying cells, as it gives chemical information from locations within the cells rather than from the cell as a whole. Glass has a large response to IR light, so it is common to fix cells to CaF₂ substrates to avoid this background; however, these substrates exhibit different interactions with the cells. It has been noticed that cells fixed at earlier timepoints after magnetic-activated cell (MAC) sorting differ in appearance from cells fixed at later timepoints. This MAC sorting process involves freeing cells from the surface (and other cells) using trypsin and having an appropriate protein that binds to the functionalized magnetic nanoparticles, allowing for magnetic separation of subpopulations within a cell line. As cells proliferate and progress through the cell cycle, the amount of genetic material, proteins, and other molecules change. This study looks at whether the molecular information from O-PTIR changes based on the timepoint of fixation of the colorectal cancer cells to the CaF₂ disks. Using machine learning, we can determine the spectral components in the samples and identify significant differences between the timepoints. By determining how the timepoint of fixing the cells impacts the molecular information obtained via O-PTIR, we can assess the influence of sample preparation procedures on assays using the sorted cells.