Using fixed fiduciary markers for stage drift correction.
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 µm x 1 µm x 1 µm 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 bio-compatible, 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 ≈ 8 nm steps.
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Sang Hak
SH
Lee
Murat
M
Baday
Marco
M
Tjioe
Paul D
PD
Simonson
Ruobing
R
Zhang
En
E
Cai
Paul R
PR
Selvin
D004867:Equipment Design; D019544:Equipment Failure Analysis; D057918:Fiducial Markers; D007089:Image Enhancement / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D036103:Nanotechnology / Q000295:instrumentation*