ATOMIC FORCE MICROSCOPY/FLUORESCENCE: Dual approach measures biomolecules with 'unprecedented' accuracy, precision

Working to learn how biological cells adhere to each other and to develop new tools to study those cells, Iowa State University (Ames, IA) researchers have developed a way to make 3D measurements of single biological molecules with "unprecedented accuracy and precision."1 The new method, called standing wave axial nanometry (SWAN), measures height to within a nanometer without custom optics or special surfaces for the samples.

SWAN involves attaching a commercial atomic force microscope (AFM) to a single-molecule fluorescence microscope: The AFM tip is positioned over a focused laser beam, creating a standing wave pattern. When a molecule treated to emit light is placed within the standing wave, its fluorescence fluctuates in a way that corresponds to the tip's distance from the molecule's surface: That distance can be compared to a marker on the surface and measured.

The researchers, using fluorescent nanospheres and single strands of DNA to test their instrument, reported measurements of a molecule's height accurate to <1 nm, and precision of repeated measurements to 3.7 nm.

In a SWAN setup, the AFM tip is positioned over a focused laser beam, creating a standing wave pattern
In a SWAN setup, the AFM tip is positioned over a focused laser beam, creating a standing wave pattern. The emission of a fluorescent molecule placed within the standing wave indicates the tip's distance from the molecule's surface.

In standing wave axial nanometry (SWAN), positioning an atomic force microscope tip over a focused laser beam excites fluorescence in a particle, whose axial position can be determined with sub-nanometer accuracy and 3.7 nm precision from the phase of the emission intensity.

1. H. Li, C-F Yen, and S. Sivasankar, Nano Lett., 12, 7, 3731–3735 (2012).

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