title: 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.
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Oxford, UK.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega Arroyo
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Oxford, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Cole
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Oxford, UK.
authors.forename: Philipp
authors.identifier: http://orcid.org/0000-0003-0136-7704
authors.initials: P
authors.lastname: Kukura
mesh_terms: D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D013997:Time Factors