Image Correlation Spectroscopy with Second Harmonic Generating Nanoparticles in Suspension and in Cells

Area of Science:

• Nanoparticle imaging and spectroscopy
• Biophysics and cellular dynamics

Background:

• Fluorescent probes suffer from photobleaching, blinking, and saturation, limiting long-term studies.
• Higher-harmonic generating nanoparticles offer high contrast and photostability for advanced spectroscopy.

Purpose of the Study:

• To apply the coherent intensity fluctuation model for studying the mobility of second harmonic generating nanoparticles.
• To develop a concise protocol for quantifying diffusion coefficients from single spectroscopy measurements.
• To demonstrate the technique's applicability in aqueous suspension and biological cells.

Main Methods:

• Utilized second harmonic generating (SHG) nanoparticles (nominally 56 nm LiNbO₃).
• Applied the coherent intensity fluctuation model for diffusion coefficient quantification.
• Performed label-free raster image correlation spectroscopy (RICS) in aqueous suspension and spatiotemporal image correlation spectroscopy (STICS) in A549 human lung carcinoma cells.

Main Results:

• A diffusion coefficient of (7.5 ± 0.3) μm²/s was measured for LiNbO₃ nanoparticles in water at room temperature.
• Significantly lower and heterogeneous diffusion coefficients were observed in A549 cells, averaging (3.7 ± 1.5) × 10^-3 μm²/s.
• The developed method allows for diffusion coefficient quantification without separate point-spread-function calibrations.

Conclusions:

• Higher-harmonic generating nanoparticles provide a robust alternative to fluorescent probes for prolonged correlation spectroscopy.
• The presented method offers a streamlined approach for measuring nanoparticle diffusion in various environments.
• Demonstrated significant differences in nanoparticle mobility between aqueous suspension and cellular environments.