DNA origami-based standards for quantitative fluorescence microscopy.
Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Jürgen J
JJ
Schmied
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Mario
M
Raab
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Carsten
C
Forthmann
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Enrico
E
Pibiri
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Bettina
B
Wünsch
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Thorben
T
Dammeyer
Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany.
Philip
P
Tinnefeld
D004247:DNA / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry*; D009690:Nucleic Acid Conformation
D004247:DNA