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Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging

Abstract

Interferometric scattering microscopy (iSCAT) is a light scattering-based imaging modality that offers a unique combination of imaging speed and precision for tracking nanoscopic labels and enables label-free optical sensing down to the single-molecule level. In contrast to fluorescence, iSCAT does not suffer from limitations associated with dye photochemistry and photophysics, or the requirement for fluorescent labeling. Here we present a protocol for constructing an iSCAT microscope from commercially available optical components and demonstrate its compatibility with simultaneously operating single-molecule, objective-type, total internal reflection fluorescence microscopy. Given an intermediate level of experience with optics and microscopy, for instance graduate-level familiarity with laser beam steering and optical components, this protocol can be completed in a time frame of 2 weeks.

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Figure 1: Comparison of iSCAT with fluorescence and darkfield microscopy.
Figure 2: Recent studies that exploit the unique capabilities of iSCAT.
Figure 3: Combined iSCAT and single-molecule TIRF experimental setup.
Figure 4: Simultaneous single-particle tracking with iSCAT and TIRF.
Figure 5: Removal of static imaging background.
Figure 6: Cage assembly for fiber coupling.
Figure 7: Microscope assembly.
Figure 8: AOD placement.
Figure 9: Concept of beam scanning pulse sequence and illumination.
Figure 10: Anticipated shot noise–limited behavior for the sample system.

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Acknowledgements

J.O.A. was supported by a scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACYT, scholar: 213546), and P.K. was supported by a European Research Council (ERC) starting grant (NanoScope). The data underpinning this report is freely available at http://ora.ox.ac.uk.

Author information

Authors and Affiliations

Authors

Contributions

J.O.A. and D.C. built the optical system and performed the experiments. P.K. supervised and conceived the protocol. J.O.A., D.C. and P.K. wrote the manuscript.

Corresponding author

Correspondence to Philipp Kukura.

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The authors declare no competing financial interests.

Supplementary information

Defocusing beam profile for an aligned microscope at step 27

For an aligned microscope and an unscanned illumination, the beam profile defocuses concentrically without displacements and exhibits high degree of radial symmetry as the sample is brought in and out of focus by displacing the axial translation stage. (AVI 441 kb)

Defocusing beam profile for a misaligned microscope at step 27

For a misaligned microscope and an unscanned illumination, the beam profile exhibits lateral displacements and poor radial symmetry as the sample is brought in and out of focus by displacing the axial translation stage. (AVI 431 kb)

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Ortega Arroyo, J., Cole, D. & Kukura, P. Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging. Nat Protoc 11, 617–633 (2016). https://doi.org/10.1038/nprot.2016.022

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