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We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.

We studied the effect of the exposure of human A549 and SH-SY5Y cell lines to aqueous solutions of organic/inorganic halide perovskites CH 3 NH 3 PbI 3 (MAPbI 3 ) and CH 3 NH 3 SnI 3 (MASnI 3 ) at the molecular level by using Fourier transform infrared microspectroscopy. We monitored the infrared spectra of some cells over a few days following exposure to the metals and observed the spectroscopic changes dominated by the appearance of a strong band at 1627 cm -1 . We used Infrared (IR) mapping to show that this change was associated with the cell itself or the cellular membrane. It is unclear whether the appearance of the 1627 cm -1 band and heavy metal exposure are related by a direct causal relationship. The spectroscopic response of exposure to MAPbI 3 and MASnI 3 was similar, indicating that it may arise from a general cellular response to stressful environmental conditions. We used 2D correlation spectroscopy (2DCOS) analysis to interpret spectroscopic changes. In a novel application of the method, we demonstrated the viability of 2DCOS for band assignment in spatially resolved spectra. We assigned the 1627 cm -1 band to the accumulation of an abundant amide or amine containing compound, while ruling out other hypotheses. We propose a few tentative assignments to specific biomolecules or classes of biomolecules, although additional biochemical characterization will be necessary to confirm such assignments., Competing Interests: Figure A1. Quantification of living cells upon exposure to MAPbI3 and MASnI3. (a,c) SH-SY5Y neuroblastoma cells; (b,d) A549 lung epithelial cells. Histograms show the average of triplicate measurements. Bars are means ± σ. The histograms show an average of at least three independent repeats. Bars are means ± σ. One-way ANOVA tests followed by Tukey-Kramer post-hoc tests were performed (non-treated vs. MAPbI3 or vs. MASnI3 treated conditions), *p < 0.01, **p < 0.005, ***p < 0.0005. Panel b is reproduced from [2] with permission from the Royal Society of Chemistry. Panels (a–d) are reproduced from [6] (I.B. doctoral thesis).

Photothermal-induced resonance (PTIR) spectroscopy and imaging with infrared light has seen increasing application in the molecular spectroscopy of biological samples. The appeal of the technique lies in its capability to provide information about IR light absorption at a spatial resolution better than that allowed by light diffraction, typically below 100 nm. In the present work, we tested the capability of the technique to perform measurements with subcellular resolution on intact eukaryotic cells, without drying or fixing. We demonstrate the possibility of obtaining PTIR images and spectra from the nucleus and multiple organelles with high resolution, better than that allowed by diffraction with infrared light. We obtain particularly strong signal from bands typically assigned to acyl lipids and proteins. We also show that while a stronger signal is obtained from some subcellular structures, other large subcellular components provide a weaker or undetectable PTIR response. The mechanism that underlies such variability in response is presently unclear. We propose and discuss different possibilities, addressing thermomechanical, geometrical, and electrical properties of the sample and the presence of cellular water, from which the difference in response may arise.