Optimized analysis for sensitive detection and analysis of single proteins via interferometric scattering microscopy

Abstract

It has been shown that interferometric detection of Rayleigh scattering (iSCAT) can reach an exquisite sensitivity for label-free detection of nano-matter, down to single proteins. The sensitivity of iSCAT detection is intrinsically limited by shot noise, which can be indefinitely improved by employing higher illumination power or longer integration times. In practice, however, a large speckle-like background and technical issues in the experimental setup limit the attainable signal-to-noise ratio. Strategies and algorithms in data analysis are, thus, crucial for extracting quantitative results from weak signals, e.g. regarding the mass (size) of the detected nano-objects or their positions. In this article, we elaborate on some algorithms for processing iSCAT data and identify some key technical as well as conceptual issues that have to be considered when recording and interpreting the data. The discussed methods and analyses are made available in the extensive python-based platform, PiSCAT 6 6 https://piscat.readthedocs.io/..

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

“…Indeed, iSCAT measurements have recently been calibrated to determine protein mass 11,12 , leading to a technology that is now also offered commercially (Refeyn Ltd., Oxford, UK). Given that the sensitivity of iSCAT is ultimately limited by shot noise 10,13,14 , one could expect to detect an arbitrarily small amount of matter if only one could collect a sufficiently large number of photons. In practice, however, the dynamics of residual background fluctuations prevents one from reaching this ideal situation 14 , and hence proteins lighter than approximately 40 kDa have not been detected 11 .…”

“…Given that the sensitivity of iSCAT is ultimately limited by shot noise 10,13,14 , one could expect to detect an arbitrarily small amount of matter if only one could collect a sufficiently large number of photons. In practice, however, the dynamics of residual background fluctuations prevents one from reaching this ideal situation 14 , and hence proteins lighter than approximately 40 kDa have not been detected 11 . In this work, we report on a substantial improvement in iSCAT detection sensitivity to Article https://doi.org/10.1038/s41592-023-01778-2 intensity of the scattered light (|E s | 2 ) becomes negligible compared with the other terms in equation (1).…”

“…1b was recorded in one frame in a short exposure time of 20 μs. Visualizing single proteins, however, requires longer integration times and an elaborate analysis to account for the speckle-like background features that are caused by coherent scattering from slight imperfections of the sample surface 14 . In brief, this analysis exploits the temporal change of the signal as a protein lands on the sensor substrate to eliminate the static background of the sample by comparing each video frame with its neighbors.…”

Decrypting Material Performance by Wide-field Femtosecond Interferometric Imaging of Energy Carrier Evolution

“…After all, the employment of a spatial filter is one of the effective strategies to enhance the image contrast without reducing the SNR. To achieve better performance in Femto-iSCAT, upgrading cameras with larger well depth and lower noise or developing new data analysis algorithms is also likely to be prospective orientations . In addition, Femto-iSCAT is sensitive to changes in the dielectric constant; thus, in principle, not only energy carriers but also molecules or ions are possible to be detected.…”

“…To achieve better performance in Femto-iSCAT, upgrading cameras with larger well depth and lower noise or developing new data analysis algorithms is also likely to be prospective orientations. 69 In addition, Femto-iSCAT is sensitive to changes in the dielectric constant; thus, in principle, not only energy carriers but also molecules 70 or ions are possible to be detected. Furthermore, Femto-iSCAT is a promising tool for unraveling ultrafast energy and carrier transfer in single particles and even single molecules, which will promote research studies on biosensing, optomechanics, heterogeneous catalysis, and solar energy harvesting.…”