Abstract
Photo-Thermal Induced Resonance (PTIR) nanospectroscopy, tuned towards amide-I absorption, was used to study the distribution of proteic material in 34 different HeLa cells, of which 18 were chemically stressed by oxidative stress with Na3AsO3. The cell nucleus was found to provide a weaker amide-I signal than the surrounding cytoplasm, while the strongest PTIR signal comes from the perinuclear region. AFM topography shows that the cells exposed to oxidative stress undergo a volume reduction with respect to the control cells, through an accumulation of the proteic material around and above the nucleus. This is confirmed by the PTIR maps of the cytoplasm, where the pixels providing a high amide-I signal were identified with a space resolution of ∼300 × 300 nm. By analyzing their distribution with two different statistical procedures we found that the probability to find protein clusters smaller than 0.6 μm in the cytoplasm of stressed HeLa cells is higher by 35% than in the control cells. These results indicate that it is possible to study proteic clustering within single cells by label-free optical nanospectroscopy.
Highlights
IntroductionOne of the most promising for biological applications is Photo-Thermal Induced Resonance (PTIR) spectroscopy, called AFM-IR nanospectroscopy because it couples to Atomic Force Microscopy (AFM) an InfraRed (IR) intense and pulsed source, like a Free Electron Laser (FEL), an Optical Parametric Oscillator (OPO) or a Quantum Cascade Laser (QCL)
Protein aggregation in cell cytoplasm is a crucial biological phenomenon
One of the most promising for biological applications is Photo-Thermal Induced Resonance (PTIR) spectroscopy, called AFM-IR nanospectroscopy because it couples to Atomic Force Microscopy (AFM) an InfraRed (IR) intense and pulsed source, like a Free Electron Laser (FEL), an Optical Parametric Oscillator (OPO) or a Quantum Cascade Laser (QCL)
Summary
One of the most promising for biological applications is Photo-Thermal Induced Resonance (PTIR) spectroscopy, called AFM-IR nanospectroscopy because it couples to Atomic Force Microscopy (AFM) an InfraRed (IR) intense and pulsed source, like a Free Electron Laser (FEL), an Optical Parametric Oscillator (OPO) or a Quantum Cascade Laser (QCL). Oxidative stress has been found to induce protein clustering in those specimens, not necessarily generating insoluble aggregates of misfolded proteins which can be detected by infrared spectroscopy through their spectral fingerprints. By analyzing the data with two different statistical procedures we obtain consistent indications of an increase of protein clustering in the stressed cells, with respect to the control ones, by about 35%. These results are encouraging from the perspective of future spectrally resolved infrared imaging directly on mutated cells that may lead to the identification of pathological inclusions in single cells
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