PiSCAT: An open source package in Python for interferometric Scattering Microscopy

iSCAT microscopy was introduced about two decades ago ([1]) and demonstrated to be the method of choice for label-free imaging and tracking of matter at nanometric scale ([2]), with a wide range of applications such as detection of gold nanoparticles, single dye molecules, viruses, and small proteins ([3]). The image of a nanoparticle in iSCAT microscopy is formed via the interference between the light scattered from the particle and a reference field which is a part of the incident laser light. The photostable scattering signal from nanoparticles allows for very long measurements at high speeds, all the way up to megahertz, limited only by the available technology, e.g. of cameras or scanners. Recording fast and long videos however, produces a large volume of data which needs to undergo several stages of computationally demanding analysis. We introduce PiSCAT as a python-based package for the analysis of variuos iSCAT measurements and related experiments. PiSCAT aims to facilitate high-performance quantitative analysis of big data and provide a generally open-access platform to enable and speed up the research in iSCAT and related communities. To facilitate the use of PiSCAT, we offer tutorials with live-code features in which we present state-of-the-art algorithms for iSCAT microscopy. These cover important educative materials in jupyter notebooks, supported with a web-based documentation page.

In this first release, we provide analysis tools for the sensitive detection of single unlabelled proteins via . wide-field iSCAT microscopy. Proteins are only a few nanometers in size with a molecular weight of a few to several hundred kDa. They were detected via iSCAT already in 2014 for small proteins down to the Bovines Serumalbumin (BSA) protein with a mass of 65 kDa ([4]). iSCAT microscopy is since employed in several more advanced applications such as real-time investigation of cellular secretion ([5], [6]) and quantitative mass spectrometry of single proteins ([7]).


The documentation webpage of PiSCAT modules can be found here.

The outputs from most of the PiSCAT localization and tracking methods are of Panda data frame type. This data structure has the ability to be easily appended/extended with more information based on different levels of analysis. The data structures containing the results of localization and tracking routines can be saved as csv, mat and HDF5 files. This helps users to work with the analyzed information using different softwares namely, MATLAB and Microsoft Excel. HDF5 is a well-known format that is readable in different programming languages and supports large, complex, heterogeneous data. HDF5 uses a “file directory” like structure that allows users to organize data within the file in structured ways and to embed metadata as well, making it self-describing.


  1. Lindfors, Kalkbrenner, et al. “Detection and spectroscopy of gold nanoparticles using supercontinuum white light confocal microscopy.” Physical review letters 93.3 (2004): 037401.

  2. Taylor, Richard W., and Vahid Sandoghdar. “Interferometric scattering microscopy: seeing single nanoparticles and molecules via rayleigh scattering.” Nano letters 19.8 (2019): 4827-4835.

  3. https://en.wikipedia.org/wiki/Interferometric_scattering_microscopy

  4. Piliarik, Marek, and Vahid Sandoghdar. “Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites.” Nature communications 5.1 (2014): 1-8.

  5. Gemeinhardt, André, et al. “Label-free imaging of single proteins secreted from living cells via iSCAT microscopy.” JoVE (Journal of Visualized Experiments) 141 (2018): e58486.

  6. McDonald, Matthew P., et al. “Visualizing single-cell secretion dynamics with single-protein sensitivity.” Nano letters 18.1 (2018): 513-519.

  7. Young, Gavin, et al. “Quantitative mass imaging of single biological macromolecules.” Science 360.6387 (2018): 423-427.

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