“O-PTIR operates in a far-field, noncontact mode, eliminating probe-related artifacts and consumables costs. This method enables the simultaneous acquisition of colocalized bright-field images, fluorescent signals from labeled nanoplastics, and submicron infrared absorption spectra, all with spatial resolutions approaching 500 nm…O-PTIR spectra are notably well-resolved, with minimal artifacts and excellent signal-to-noise ratios.”
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Reporting in ACS Applied Materials & Interfaces, researchers at the Institute of Nuclear Physics Polish Academy of Sciences and Singapore Synchrotron Light Source demonstrated the first reliable detection and spatial mapping of nanoplastics within individual human cells at submicron and nanoscale resolutions. The detection and intracellular tracking of nanoplastics in human cells remain critical challenges in understanding their biological impact. Nanoplastics, defined as plastic particles smaller than 1 μm, can traverse biological barriers including the blood-brain barrier and accumulate in organs and tissues. While fluorescence microscopy offers localization insights for labeled nanoparticles, it lacks chemical specificity and suffers from photobleaching. The team addressed this limitation using multimodal vibrational spectroscopy combining fluorescence imaging, Raman spectroscopy, FT-IR, AFM-IR, and O-PTIR to detect and characterize fluorescently labeled 160 nm polystyrene nanoparticles (PS-NPs) within fibroblast and glioblastoma cell lines.
MTS viability assays confirmed that PS-NP concentrations (0.005-0.02 mg/mL) did not substantially compromise cell viability in either cell line, aligning with the objective of detecting rather than inducing cytotoxicity. Three-dimensional Raman imaging confirmed intracellular localization using the PS-NP marker band at ~1601 cm⁻¹, revealing nanoparticles predominantly surrounding the nucleus in a characteristic perinuclear ring pattern. However, conventional FT-IR microspectroscopy failed to detect PS-NPs within single cells due to spatial resolution constraints imposed by the diffraction limit. Numerical spectral mixing simulations established the threshold concentration at which PS-NP detection would be feasible.
AFM-IR overcame the limitations of spatial resolution by combining infrared absorption with atomic force microscopy to achieve nanoscale spatial resolution (tens of nanometers). The technique successfully identified characteristic PS-NP spectral signatures at ~1492 cm⁻¹ in both cell lines, with intensity variations reflecting nanoparticle distribution and aggregation state. AFM-IR provided both chemical spectra and topographical information, enabling visualization of PS-NP localization within subcellular compartments. O-PTIR complemented AFM-IR by operating in a noncontact mode, eliminating probe-related artifacts while achieving spatial resolutions approaching 500 nm. The technique enabled simultaneous acquisition of bright-field cellular images, fluorescent signals from labeled nanoplastics, and submicron infrared absorption spectra. O-PTIR successfully detected the ~1492 cm⁻¹ aromatic C═C stretching vibration marker band, with spatial distribution strongly correlating with fluorescence microscopy results indicating perinuclear nanoparticle accumulation.
Direct comparison revealed that both AFM-IR and O-PTIR successfully confirmed intracellular PS-NP presence and enabled chemical distribution mapping at single-cell resolution. AFM-IR offered superior spatial resolution (tens of nanometers) and detailed topographical mapping, making it ideal for nanoscale analysis. O-PTIR provided noncontact submicron resolution, operational simplicity, excellent spectral fidelity with minimal artifacts, and exceptional signal-to-noise ratios. Hyperspectral O-PTIR imaging demonstrated the ability to collect 81 spectra over a 4×4 μm² area with 0.5 μm step size, exploiting wavelength-independent submicron capabilities. The findings establish photothermal-based IR spectroscopy as powerful tools for precise, noninvasive detection of nanoplastics within individual human cells, laying groundwork for future investigations into cellular fate and biological impact at previously unattainable resolution scales.
Authors:
Ewa Pięta, Natalia Piergies, Karolina Chrabąszcz, Agnieszka Banas, Krzysztof Banaś, Michael K. F. Lo, Agnieszka Panek, Wojciech M. Kwiatek, Mark B. H. Breese, and Katarzyna Pogoda
First Author Institution: Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland
