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Synchrotron photothermal IR nanospectroscopy of macrophages drug-induced phospholipidosis

DOI: 10.1021/acs.analchem.9b05759 DOI Help

Authors: Ka Lung Andrew Chan (King's College London) , Ioannis Lekkas (Diamond Light Source) , Mark D. Frogley (Diamond Light Source) , Gianfelice Cinque (Diamond Light Source) , Ali Altharawi (King's College London) , Gianluca Bello (University of Natural Resources and Life Sciences) , Lea Ann Dailey (University of Vienna)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Analytical Chemistry

State: Published (Approved)
Published: May 2020
Diamond Proposal Number(s): 21061

Abstract: Synchrotron resonance-enhanced infrared-atomic force microscopy (RE-AFM-IR) is a near-field photothermal vibrational nanoprobe developed at the Diamond Light Source (DLS), capable of measuring mid-infrared absorption spectra with spatial resolution around 100 nm. The present study reports a first application of synchrotron RE-AFM-IR to interrogate biological soft matter at subcellular level, in this case on a cellular model of drug-induced phospholipidosis (DIPL). J774A-1 macrophages were exposed to amiodarone (10 ┬ÁM) or medium for 24 hours and chemically fixed. AFM topography maps revealed amiodarone-treated cells with enlarged cytoplasm and very thin regions corresponding to collapsed vesicles. IR maps of the whole cell were analysed by exploiting the RE-AFM-IR overall signal, i.e. the integrated RE-AFM-IR signal amplitude versus AFM-derived cell thickness, also on lateral resolution around 100 nm. Results shown that vibrational band assignment was possible and all characteristic peaks for lipids, proteins and DNA/RNA were identified. Both peak ratio and unsupervised chemometric analysis of RE-AFM-IR nanospectra generated from the nuclear and perinuclear regions of untreated and amiodarone-treated cells showed that the perinuclear region (i.e. cytoplasm) of amiodarone-treated cells had significantly elevated band intensities in the regions corresponding to phosphate and carbonyl groups, indicating detection of phospholipid-rich inclusion bodies typical for cells with DIPL. The results of this study are of importance to demonstrate not only the applicability of Synchrotron RE-AFM-IR to soft biological matters with subcellular spatial resolution, but also that the spectral information gathered from an individual sub-micron sample volume enables chemometric identification of treatment and biochemical differences between mammalian cells.

Journal Keywords: RE-AFM-IR; Synchrotron IR; photothermal nanospectroscopy; nanoFTIR; PTIR

Subject Areas: Chemistry, Biology and Bio-materials, Technique Development

Instruments: B22-Multimode InfraRed imaging And Microspectroscopy