期刊
NATURE PROTOCOLS
卷 9, 期 10, 页码 2317-2328出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.155
关键词
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资金
- University of Wisconsin-Madison
- Wisconsin Alumni Research Foundation (WARF)
- Department of Biochemistry
- US National Institute of Health (NIH) [R00 GM086471]
- US Department of Agriculture [WIS 01625]
- NIH [R01 GM81648, GM38660]
- G. Harold and Leila Y. Mathers Foundation
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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