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Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope


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.

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Figure 1: An example of the use of the mmTIRFM in a CoSMoS experiment to study spliceosome assembly.
Figure 2: A general schematic of an objective-based mmTIRFM.
Figure 3: The components of the mmTIRF system and their assembly.
Figure 4: Micromirror platforms and their mounting underneath the objective.
Figure 5
Figure 6: The alignment tool used for orientation of the excitation and emission paths through the objective.
Figure 7: The components and layout of the emission path optics.


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We acknowledge support from startup funding from the University of Wisconsin-Madison, Wisconsin Alumni Research Foundation (WARF) and the Department of Biochemistry. A.A.H. is also supported by a K99/R00 career transition award from the US National Institute of Health (NIH) (R00 GM086471) and is a Beckman Young Investigator of the Arnold and Mabel Beckman Foundation. J.L. and A.A.H. are supported by a Hatch Act Formula Fund from the US Department of Agriculture (WIS 01625). L.J.F. is supported by NIH grant R01 GM81648 (to J. Gelles, Brandeis University) and a grant from the G. Harold and Leila Y. Mathers Foundation. Funds for purchase of many of the components of the mmTIRF system described here were generously supplied by Professor Robert Landick (University of Wisconsin-Madison) and an NIH administrative supplement award (GM38660).

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J.L. constructed the microscope with assistance from A.A.H.; M.K. integrated the microscope hardware with the image acquisition software; A.A.H., J.L., E.A.D., W.O. and J.F.M. designed and constructed the mmTIRF system; L.J.F. provided crucial advice; and A.A.H. and J.L. wrote the manuscript with input from the other authors.

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Correspondence to Aaron A Hoskins.

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Competing interests

M.K., E.A.D., W.O. and J.F.M. are employees of Mad City Labs.

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Larson, J., Kirk, M., Drier, E. et al. Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9, 2317–2328 (2014).

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