iSCAT

Interferometric Scattering Microscopy (iSCAT) is a label-free optical microscopy technique that detects light scattered by nanoscale objects by interfering it with a reference light field. The method enables visualization and tracking of objects smaller than the diffraction limit, including single nanoparticles and biomolecules, without fluorescent labels.

Principle: iSCAT relies on coherent illumination of the sample. The light scattered by the object interferes with a reference field, often the light reflected from the substrate. The detected intensity contains an interference term that scales with the particle’s complex scattering amplitude. This interferometric gain makes weak scattering detectable and allows, with calibration, semi-quantitative estimates of size and polarizability. Phase stability and careful geometry are important for reliable measurements.

Instrumentation: A typical setup uses a standard optical microscope with a coherent light source (laser or narrow-band illumination) and a high numerical aperture objective. A camera records interferometric contrast, and digital processing improves signal-to-noise and enables real-time tracking. The geometry can be based on reflection/refraction configurations, sometimes with oblique illumination, to optimize the reference field and background suppression.

Applications and advantages: iSCAT is used for label-free tracking and imaging of nanoparticles, vesicles, viral particles, and biomolecules, in solution or on surfaces. It provides high temporal resolution and the potential for quantitative measurements of diffusion, binding kinetics, and transport, without the need for fluorescent labels. It is especially useful for fast dynamics and for samples where labeling is undesirable or perturbative.

Limitations: The technique is sensitive to background and surface roughness, requiring careful calibration and optical stability. Interpreting contrast can be challenging in heterogeneous or thick samples, and specialized data processing is often needed. Ongoing development seeks to improve background rejection and quantitative accuracy.