Contrast-Enhancing Spatial–Frequency Deconvolution-Aided Interferometric Scattering Microscopy (iSCAT)

Interferometric scattering microscopy (iSCAT) is widely used for label-free tracking of nanoparticles and single molecules. However, its ability to identify small molecules is limited by low imaging contrast blurred with noise. Frame-averaging methods are widely used for reducing background noise but require hundreds of frames to produce a single frame as a trade-off. To address this, we applied a spatial–frequency domain deconvolution algorithm to suppress background noise and amplify the signal for each frame, achieving an improvement of ∼ 3-fold without hardware modification. This enhancement is achieved by compensating for missing information within the optical transfer function (OTF) boundary, while high-frequency components (noise) beyond this boundary are filtered. The resulting deconvolution process provides linear signal amplification, making it ideal for quantitative analysis in mass photometry. Additionally, the localization error is reduced by 20%. Comparisons with traditional denoising algorithms revealed that these methods often extract the side lobes. In contrast, our deconvolution approach preserves signal integrity while enhancing sensitivity. This work highlights the potential of image processing techniques to significantly improve the detection sensitivity of iSCAT for small molecule analysis.