FTIR Imaging with Novel ZnS Hemispheres for Studying Phospholipidosis in Live Macrophages at Subcellular Level

Abstract

The respiratory system plays crucial roles in gas exchange and defence against airborne pathogens. Alveolar macrophages (AMs), vital in this defence within the respiratory tree, undergo morphological and biochemical changes when exposed to xenobiotics, forming lipid vacuoles in their cytoplasm. Pathologists commonly refer to these changes as foamy macrophages (FMs). This response presents challenges for developing new inhaled medicines. In preclinical studies, the presence of FMs in rats can delay development, restrict dosages for clinical evaluation, or even lead to non-approval due to toxicity concerns. While not all formed FMs are recruited to fight infection, they can also be formed as an adaptive (reversible) response. A simple and affordable test to distinguish between adaptive and adverse responses has not been established yet. In addressing this challenge, we investigated if FTIR imaging or microscopy can be used to distinguish these responses by developing and testing the methods against a model utilising the J774A.1 murine macrophage-like cell line. A novel hemispherical-optical substrate device, capable of imaging at the single-cell level while maintaining cell viability and providing high-quality spectra with enhanced spatial resolution, was used. Various phospholipid-inducing drugs, such as amiodarone (an adverse-response inducer), fluticasone (an adaptive-response inducer), and salbutamol (a negative control), were used to demonstrate the developed measurement approach. The demountable liquid-sample holder contains two hemispheres of zinc sulphide with live cells sandwiched between them and a 6-μm spacer to minimise water interference. This configuration allowed for the removal of chromatic aberration and increased the spatial resolution of the measurement to produce spectra with clear and easily analysable hydrocarbon and fingerprint regions that require minimal data processing. Furthermore, the system exhibited the capability to detect changes in the DNA side chain and the carbonyl ester bands at ≈1714 and ≈1745 cm-1, respectively. Subsequently, the system was further developed into a dynamic flow configuration (flow-cell), enabling real-time measurement of subcellular responses to chemical stimuli while preserving cell viability for extended durations. Validation and optimisation of the developed flow-cell were conducted using a standard and a synchrotron-based FTIR imaging microscope (SR-FTIR), acquiring detailed single-cell and subcellular components. The incorporation of a focal plane array detector and SR-FTIR with the novel device was also demonstrated for the first time, providing an improvement in image acquisition time and identification of spectral features. Finally, Raman microspectroscopy was also employed as a complementary method. Both systems, FTIR and Raman, demonstrated the increase in lipid content following treatment with amiodarone and fluticasone, specifically the CH2 asymmetric and symmetric bands and olefinic CH stretching at the high wavenumber region. There were also increases in the carbonyl ester band at ≈1740 cm-1 and the CH2 bending band in the low wavenumber region. Amiodarone presented with clear, drastic changes, whereas fluticasone exhibited similar spectral features but to a lesser extent. The results suggest that FTIR has the potential to become a specific analytical tool, with a low running cost, to distinguish the adaptive and adverse response from macrophages in preclinical drug toxicity screening.