Synchrotron InfraRed Nanospectroscopy has been used for the first time to measure biomolecular changes caused by a drug (amiodarone) in human cells (macrophages) and localized at 100 nanometre scale, i.e. two orders of magnitude smaller than the IR wavelength used as probe.

This was achieved at the <span style="color: #ff6600;"><a style="color: #ff6600;" href="https://pubs.acs.org/doi/10.1021/acs.analchem.9b05759">Multimode InfraRed Imaging</a></span> and <span style="color: #ff6600;"><a style="color: #ff6600;" href="https://pubs.acs.org/doi/10.1021/acs.analchem.9b05759">Micro-Spectroscopy (MIRIAM) beamline (B22) in Diamond Light Source, the UK's national synchrotron facility.</a></span>

This is a major scientific effect in Life Sciences shared by an Worldwide team, as a collaborative beamtime among the researchers from the School of Cancer and Pharmaceutical Science at Kings College London, the Department of Pharmaceutical Technology and Bio-pharmacy in University of Vienna, and the scientists of their <span style="color: #ff6600;"><a style="color: #ff6600;" href="https://pubs.acs.org/doi/10.1021/acs.analchem.9b05759">MIRIAM B22 beamline in Diamond.</a></span>

<em>This is remarkable because the determination of lipid content in vacuoles is crucial in the study of DIPL. This will have high impacts on the development of inhaled medicines whereby DIPL is one of the key indications of adverse response from the body to foreign particles."-</em>Dr Andrew Chan, Principal Investigator, King's College London

Their latest paper, now published in Analytical Chemistry, is titled"Synchrotron photothermal IR Nanospectroscopy of macrophages drug-induced phospholipidosis" 10.1021/acs.analchem.9b05759 It outlinesthe use of this so-called Resonance Improved InfraRed Atomic Force Microscopy (RE AFM IR) by Synchrotron Radiation, to interrogate biological thing at the subcellular level, in this case a cellular model of drug-induced phospholipidosis (DIPL).

Rather than the traditional procedure to evaluate DIPL - i.e. visual affirmation by electron microscopy of these lipid bodies or the use of fluorescence labeling technique - they used IR broadband illumination by Diamond synchrotron together with AFM detection to achieve both molecular specificity and enhanced spatial resolution needed to localize metabolic alterations inside the cell.

Dr Andrew Chan of King's College London as principal researcher explains,"The model study according to J774A-1 macrophages exposed/not subjected to amiodarone has clearly demonstrated that RE AFM IR with synchrotron radiation is effective at extracting local molecular information from small organelles inside a single cell within an label-free manner."

AFM topography maps revealed amiodarone-treated cells had expanded cytoplasm, and sparse areas of collapsed vesicles. The Infra Red (IR) maps of the whole cell were analysed by exploiting the IR overall signal versus AFM-derived cell depth, also on lateral resolution about 100 nm. Vibrational band assignment of this nanospectra was possible too: all characteristic peaks for lipids, proteins, and DNA/RNA were also identified.

Also, both band ratio and unsupervised chemometric analysis of Synchrotron IR nanospectra in the nuclear and perinuclear regions of the cells revealed that the cytoplasm of both amiodarone-treated cells had substantially elevated group intensities in the areas corresponding to phosphate and carbonyl groups, signaling detection of phospholipid-rich inclusion bodies typical for cells using DIPL.
Principle Beamline Scientist in the MIRIAM beamline at Diamond and one of the job's authors, Dr Gianfelice Cinque comments:"Our experiment is - to my knowledge - a world first by Synchrotron photothermal IR Nano spectroscopy in life sciences, also demonstrated that photothermal IR Nano spectroscopy can successfully scan across mammalian cells and reveal the inner molecular fingerprint via the complete IR spectrum, as a result of Synchrotron IR broadband coverage."

<span style="color: #ff6600;"><a style="color: #ff6600;" href="https://doi.org/10.1021/acs.analchem.9b05759">He explained that the mobile model system and the drug treatment exemplified the method capability by spatially colocalizing morphology and biochemistry in subcellular scale.</a> </span>What was notable was that the nanospectra caliber attained was such that average vibrational features observed by IR microscopy on biological cells were obviously recorded, but also for the first time in the nanoscale, supplying subcellular biochemical information in a label-free manner.

<span style="color: #ff6600;"><a style="color: #ff6600;" href="https://doi.org/10.1021/acs.analchem.9b05759">He adds;" This achievement has been the end of a long experimental endeavor by the IR beamline B22 group of Diamond - particularly the expert work by Dr Mark Frogley and Dr Ioannis Lekkas."</a></span>

He proceeded to explain that the MIRIAM beamline (B22) excellence in Synchrotron IR Nano spectroscopy - i.e. Synchrotron RE-AFM-IR spectroscopy - offers exceptional chemical and morphological penetration at sub-wavelength or 100 nm resolution across a variety of real life study especially in soft matter, for example microplastic influence in living tissue, antimicrobial surface happenings, microfossil and biogeology at submicron scale, organic microelectronics analysis, microcomposite substances and mesostructures.

More research capability will be offered shortly at the MIRIAM beamline B22, as a brand new nanoIR end channel is likely from mid 2021. Past the present expertise in Synchrotron IR photothermal Nanospectroscopy, the upgrade will allow new techniques (e.g. AFM IR in tapping mode), and crucially complement them with IR scattering scanning optical microscopy (s-SNOM), pushing the spatial resolution even farther in the 10s nanometre scale.
<div class="content-src-label"><strong>Source:</strong></div>
<div class="content-src-value">

<span style="color: #ff6600;"><a style="color: #ff6600;" href="https://www.diamond.ac.uk/Home" target="_blank" rel="noopener noreferrer">Diamond Light Source Ltd</a></span>

</div>
<div class="content-src-label"><strong>Journal reference:</strong></div>
<div class="content-src-value">

Chan, K. L. A., <em>et al</em>. (2020) Synchrotron Photothermal Infrared Nanospectroscopy of Drug-Induced Phospholipidosis in Macrophages. <em>American Chemical Society</em>.<span style="color: #ff6600;"> <a style="color: #ff6600;" href="https://doi.org/10.1021/acs.analchem.9b05759" target="_blank" rel="noopener noreferrer">doi.org/10.1021/acs.analchem.9b05759.</a></span>

</div>