Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa

Interferometric scattering (iSCAT) microscopy is a label-free optical method capable of detecting single proteins, localizing their binding positions with nanometer precision, and measuring their mass. In the ideal case, iSCAT is limited by shot noise such that collection of more photons should extend its detection sensitivity to biomolecules of arbitrarily low mass. However, a number of technical noise sources combined with speckle-like background fluctuations have restricted the detection limit in iSCAT. Here, we show that an unsupervised machine learning isolation forest algorithm for anomaly detection pushes the mass sensitivity limit by a factor of 4 to below 10 kDa. We implement this scheme both with a user-defined feature matrix and a self-supervised FastDVDNet and validate our results with correlative fluorescence images recorded in total internal reflection mode. Our work opens the door to optical investigations of small traces of biomolecules and disease markers such as α-synuclein, chemokines and cytokines.