Using fixed fiduciary markers for stage drift correction. Academic Article uri icon

Overview

abstract

  • 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.

publication date

  • May 21, 2012

Research

keywords

  • Fiducial Markers
  • Image Enhancement
  • Microscopy, Fluorescence
  • Nanotechnology

Identity

PubMed Central ID

  • PMC3482917

Scopus Document Identifier

  • 84861501724

Digital Object Identifier (DOI)

  • 10.1039/c0an00144a

PubMed ID

  • 22714205

Additional Document Info

volume

  • 20

issue

  • 11