title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.
abstract: Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.
authors.affiliation: 1] Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA. [2] Biophysics Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Larson
authors.affiliation: Mad City Labs Inc., Madison, Wisconsin, USA.
authors.forename: Matt
authors.initials: M
authors.lastname: Kirk
authors.affiliation: Mad City Labs Inc., Madison, Wisconsin, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Drier
authors.affiliation: Mad City Labs Inc., Madison, Wisconsin, USA.
authors.forename: William
authors.initials: W
authors.lastname: O'Brien
authors.affiliation: Mad City Labs Inc., Madison, Wisconsin, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: MacKay
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Hoskins
mesh_terms: D004867:Equipment Design; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods