Journal
OPTICS EXPRESS
Volume 20, Issue 11, Pages 12177-12183Publisher
Optica Publishing Group
DOI: 10.1364/OE.20.012177
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Funding
- NIH [GM068625]
- NSF [DBI- 02-15869, EAGER 0968976, 082265]
- Direct For Biological Sciences
- Div Of Biological Infrastructure [1063188] Funding Source: National Science Foundation
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0968976] Funding Source: National Science Foundation
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To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 mu m x 1 mu m x 1 mu m in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are biocompatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and < 17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with approximate to 8 nm steps. (C) 2012 Optical Society of America
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