Implementation of Liquid Cell System of Measurement for Synchrotron Based IR Microanalysis of Live Biological Cells in Cell Biology and Biotechnology

UoM administered thesis: Phd

  • Authors:
  • James Doherty

Abstract

Fourier transform infrared spectroscopy (FTIR) and microscopy (FT-IRMS) are well-established as tools for the study of biological cells. Studies of chemically-fixed cells using infrared (IR) have been able to yield significant information regarding cell morphology, disease development and drug-cell interactions. However, such studies are limited by fixation effects which introduce a range of spectral variations, limiting the interpretation of IR spectra of fixed cells. It is therefore desirable to study living cells in real time, particularly as cells are undergoing response to an external stimulus. This does, however, present a number of challenges. Maintaining live cells requires an aqueous sample environment. Water is, however, a very strong absorber of IR light, producing a spectrum which dwarfs the biochemical signals in the sample and makes the extraction of good-quality spectra with high signal-to-noise (S/N) extremely challenging. The brilliance of synchrotron radiation (SR), combined with development of an in-house water correction procedure performing a least-squares fit with a (Matrigel) reference spectrum, has been able to generate high-quality spectra while retaining a sufficient level of spectral detail to analyse relative subtle biochemical changes. Wildtype and drug-resistant strains of renal cell carcinoma (RCC) have been treated with different chemotherapy agents and formalin-fixed. Analysis of fixed, dried samples using both single-point SR IR microscopy and bench-top focal plane array (FPA) imaging has identified spectral features linked to efflux or similar drug-resistance mechanisms, opening the possibility of being able to identify drug resistance in the early stages of treatment. These results were able to be replicated using fixed cells suspended in phosphate-buffered saline (PBS), providing validation of the water correction algorithm. Two live cell studies were performed in a static aqueous sample holder. K562 acute myeloid leukaemia (AML) cells were treated with two novel chemotherapy agents, and differences were identified in the resulting spectra that could be directly linked to the mode of action of the two compounds. This analysis made use of DNA bands that are only accessible when measuring hydrated cells. The second study used a wildtype and drug-resistant strain of PEO1 ovarian cancer cells, and analysed their response to a novel agent, showing differences in the measured spectra between wildtype and resistance samples. Static-mode live cell studies have been able to provide significant information, but are limited by the survival time of the samples. A customised commercial liquid sample holder has undergone simple and cost-effective modifications to produce a dynamic flow system for live cell measurements, potentially up to and beyond 24 hours. This includes the novel implementation of a hydrophobic barrier to direct and control flow. Trypan Blue staining has confirmed cell viability up to at least 24 hours, and two biological studies have been performed to demonstrate different properties of the system. Measuring samples at different temperatures has proved that the system is capable of inducing and simultaneously measuring cellular changes, while a study of deuterated palmitic acid uptake has shown that a sample can be maintained for long enough to acquire spectra of a large number of cells.

Details

Original languageEnglish
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Award date1 Aug 2019