Nanoscale resolved Infrared spectroscopy of Living Cells in Water
ORAL
Abstract
Nanoscale-resolved infrared imaging and spectroscopy enable the investigation of chemical and structural properties of biological specimens with unprecedented spatial resolution and sensitivity—without the need for perturbative fluorescent labeling [1–3]. Infrared nanospectroscopy (nano-FTIR) combines the high spatial resolution of atomic force microscopy (AFM) with the chemical specificity of infrared spectroscopy, thereby overcoming the diffraction limit of light and achieving wavelength-independent spatial resolution down to 10–20 nm [3].
A major challenge in applying nano-FTIR to living biological specimens in aqueous environments is the strong infrared absorption of water and the risk of tip contamination. We present a novel biocompatible flow cell design based on an ultrathin (10 nm) free-standing silicon nitride (SiN) membrane. This membrane allows the nano-FTIR tip to probe samples through it under ambient conditions, while the tip-confined infrared near-field penetrates the membrane with minimal attenuation. The design prevents liquid evaporation, oxidative damage, and mechanical distortion of the specimen.
We demonstrate the flow cell’s applicability by imaging living E. coli bacteria in nutrient medium and A549 cancer cells during division and migration. The cells exhibit rich subcellular morphology and adhesion dynamics, resolved at 150 nm spatial resolution. nano-FTIR spectra reveal local distributions of water, proteins, and lipids within ~100 nm beneath the membrane, verified through polymer sphere calibration and model calculations [4].
[1] M. Brehm et al., Nano Lett. 6, 1307 (2006)
[2] S. Gamage et al., PLoS ONE 13, e0199112 (2018)
[3] F. Keilmann, R. Hillenbrand, Phil. Trans. R. Soc. A 362, 787–805 (2004)
[4] K. J. Kaltenecker et al., Sci. Rep. 11, 21860 (2021)
A major challenge in applying nano-FTIR to living biological specimens in aqueous environments is the strong infrared absorption of water and the risk of tip contamination. We present a novel biocompatible flow cell design based on an ultrathin (10 nm) free-standing silicon nitride (SiN) membrane. This membrane allows the nano-FTIR tip to probe samples through it under ambient conditions, while the tip-confined infrared near-field penetrates the membrane with minimal attenuation. The design prevents liquid evaporation, oxidative damage, and mechanical distortion of the specimen.
We demonstrate the flow cell’s applicability by imaging living E. coli bacteria in nutrient medium and A549 cancer cells during division and migration. The cells exhibit rich subcellular morphology and adhesion dynamics, resolved at 150 nm spatial resolution. nano-FTIR spectra reveal local distributions of water, proteins, and lipids within ~100 nm beneath the membrane, verified through polymer sphere calibration and model calculations [4].
[1] M. Brehm et al., Nano Lett. 6, 1307 (2006)
[2] S. Gamage et al., PLoS ONE 13, e0199112 (2018)
[3] F. Keilmann, R. Hillenbrand, Phil. Trans. R. Soc. A 362, 787–805 (2004)
[4] K. J. Kaltenecker et al., Sci. Rep. 11, 21860 (2021)
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Publication: K. J. Kaltenecker et al., Sci. Rep. 11, 21860 (2021)
Presenters
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Tobias Gokus
- attocube Systems GmbH