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Instruments / Technical Area	Publication Details	Published	Actions
B22-Multimode InfraRed imaging And Microspectroscopy	Synchrotron photothermal IR nanospectroscopy of macrophages drug-induced phospholipidosis Ka Lung Andrew Chan , Ioannis Lekkas , Mark D. Frogley , Gianfelice Cinque , Ali Altharawi , Gianluca Bello , Lea Ann Dailey Analytical Chemistry DOI: 10.1021/acs.analchem.9b05759 Diamond Proposal Number(s): [21061] Show Abstract ▼ 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 µM) 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.	May 2020
B22-Multimode InfraRed imaging And Microspectroscopy	Transmission Fourier Transform Infrared (FT-IR) imaging, mapping, and synchrotron scanning microscopy with ZnS hemispheres on living mammalian cells at sub-cellular resolution Ka Lung Andrew Chan , Ali Altharawi , Pedro L. Fale , Cai Li Song , Sergei G. Kazarian , Gianfelice Cinque , Valerie Untereiner , Ganesh Sockalingum Applied Spectroscopy DOI: 10.1177/0003702819898275 Diamond Proposal Number(s): [17501] Show Abstract ▼ Abstract: Fourier transform infrared (FT-IR) spectroscopic imaging and microscopy of single living cells are established label-free technique for the study of cell biology. The constant driver to improve the spatial resolution of the technique is due to the diffraction limit given by IR wavelength making subcellular study challenging. Recently we have reported, with the use of a prototype ZnS transmission cell made of two hemispheres, that the spatial resolution is improved by the factor of the refractive index of ZnS, achieving a λ/2.7 spatial resolution using the synchrotron– IR microscopy with a 36x objective with numerical aperture (NA) of 0.5. To refine and to demonstrate that the ZnS hemisphere transmission device can be translated to standard bench-top FT-IR imaging systems, we have, in this work, modified the device to achieve a more precise path length, which has improved the spectral quality of the living cells, and showed for the first time that the device can be applied to study live cells with three different bench-top FT-IR imaging systems. We applied focal plane array (FPA) imaging, linear array, and a synchrotron single point scanning method and demonstrated that in all cases, subcellular details of individual living cells can be obtained. Results have shown that imaging with the FPA detector can measure the largest area in a given time whilst measurements from the scanning methods produced a smoother image. Synchrotron single point mapping produced the best quality image and has the flexibility to introduce over sampling to produce images of cells with great details, but it is time consuming in scanning mode. In summary, this work has demonstrated that the ZnS hemispheres can be applied in all three spectroscopic approaches to improve the spatial resolution without any modification to the existing microscopes.	Feb 2020

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