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/..
Highlights
The most common configuration of interferometric scattering (iSCAT) operates in the widefield reflection mode, where the light reflected from the sample interface acts as the reference field Er, and Es is collected via the same microscope objective that delivers the illumination
We propose and employ the Difference of Gaussian (DoG) [34, 35] for the optimal analysis of low-contrast iSCAT signals using only the main lobe of the iSCAT point-spread function (iPSF)
We find that DoG offers a high performance for detecting iPSFs with low signal-to-noise ratio (SNR), localizing the center, and estimating the iPSF size, all at the same time
Summary
In iSCAT, one embraces the illumination field instead of avoiding it, as is done in dark-field microscopy. Er, Ir = |Er|2, Es, and Is = |Es|2 denote the electric fields and intensities of the reference and the scattered light on the detector, respectively. The most common configuration of iSCAT operates in the widefield reflection mode, where the light reflected from the sample interface acts as the reference field Er, and Es is collected via the same microscope objective that delivers the illumination. The iSCAT point-spread function (iPSF) of a wide-field setup results from the interference of a quasi-spherical wave emitted by the nanoparticle under study and a quasi-plane wave of the reference field [14]. A cross section (see red curve) shows that an iPSF in a wide-field setup contains several interference rings, the details of which depend on the focusing parameters and the exact particle position along the optical axis. We remark that a successful iSCAT measurement requires various technical choices that we do not discuss in this work, but they can be found in [13, 16, 17]
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