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bioRxiv · 10.64898/2025.12.28.696748

Cell-in-Water: measurements using Optical Photothermal Infrared spectroscopy

Korona, W.·Nowakowska, A.·Pieczara, A.·Orzechowska, B. M.·Unger, M.·Baranska, M.
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Abstract

Conventional Fourier transform infrared absorption spectroscopy (FT-IR) allows for measurements of spatial resolution comparable to a whole cell size, which are performed on dried cells to avoid water interference. However, such conditions can disrupt the native morphology and chemical composition of cells, whilst not providing for crucial sub-cellular biochemical detail. Pump-probe Optical Photothermal Infrared (O-PTIR) Microscopy offers high-resolution measurements of cells, but the challenge of measuring cells without disturbing their morphology remains. To overcome this, we developed a protocol enabling O-PTIR measurements of cells in aqueous environments using a silicone-sealed microchamber, which allows imaging of live cells in 2 {micro}l of buffer for up to 4 hours. Analysis of O-PTIR data obtained from hydrated and dehydrated cells confirms that hydrated conditions better preserve subcellular organization, as evidenced by secondary structure changes of proteins. We also propose a step-by-step algorithm that can improve image quality through ratiometric analysis, histogram assessment, and the application of RGB overlays. This approach allows for visualization of multiple subcellular components, such as the nucleus or lipid droplets. Trough the combination of "sandwich" sample configuration and O-PTIR, we offer a promising strategy to advance O-PTIR measurements of cells in water, which have long been considered a major challenge. A direct comparison of O-PTIR with Raman microscopy highlights the complementarity of these two techniques and similar spatial resolution.

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Korona, W., Nowakowska, A., Pieczara, A., Orzechowska, B. M., Unger, M., Baranska, M.. 2025-12-28. Cell-in-Water: measurements using Optical Photothermal Infrared spectroscopy. https://doi.org/10.64898/2025.12.28.696748

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