Dynamic Single Particle Measurements with a Compact, High Throughput Iscat-Tirf Setup
BIOPHYSICAL JOURNAL(2024)
摘要
Interferometric scattering microscopy (iSCAT) enables label-free, prolonged and high framerate timelapse acquisition and is thus an ideal technique for single particle tracking. typically, iSCAT setups employ scanning illumination schemes to achieve uniform sample illumination. However, this implementation limits the maximum achievable field of view (FoV), while increasing hardware requirements and setup size. To overcome these disadvantages, we developed a scanning-free interferometric scattering microscopy setup in combination with an objective based total internal reflection microscopy (TIRF) channel. We demonstrate that it is possible to achieve a large (60 μm × 60 μm) uniformly illuminated FoV with a passive refractive optical element in the iSCAT illumination path and record images with a time resolution of 300 μs. This setup has the additional advantage of a compact footprint (<1sqm). We also made available a configuration for an open-source hardware control software (ImSwitch), to handily control the setup. We show that we can detect single particles with a uniform contrast and a <10 nm localization precision throughout the FoV, and track single gold nanoparticles (<40 nm in diameter) with a dynamic localization precision <20 nm for prolonged time, also thanks to a focus lock channel. The instrument can perform quantitative single molecule measurements as single particle tracking and imaging fluorescence correlation spectroscopy. We demonstrate and compare these measurements on different model membrane systems. For its several advantages, this illumination-detection scheme for iSCAT has the potential to expand the reach of this imaging method, and to drive further applications of this technique in Biology and Biophysics.
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