Using fixed fiduciary markers for stage drift correction

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.

Resolution limits to object tracking with subpixel accuracy

“…The use of geometrical objects permits smart interpolation in the spatial domain, and thus subpixel accuracy can be achieved. However, we could not find in the literature resolution enhancements of greater than three orders of magnitude [3][4][5].…”

“…For example, setting ε 0.25 implies a subpixel array of 4 × 4 elements. The pair (3.25, 5.75) represents a dot located at the position (2,4) inside pixel (3,5). Defining the object in this way ensures that, statistically, the spots in the image cloud are located at different locations in different pixels.…”

Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm

“…A common approach for drift measurement is by introducing suitable fiducial markers (normally small fluorescence beads) into the sample. Assuming that the markers are stationary with respect to the target structure, the sample drift can be measured by tracking the marker positions [11,12]. However, this approach needs additional sample preparation efforts.…”

Test Samples for Optimizing STORM Super-Resolution Microscopy

“…Even if fiducial markers cannot be incorporated into every experiment they can, at least, be used to characterize a microscope system, its environment and better understand operational considerations such as how much time is required to reach thermal equilibrium after start up. As well as correcting for lateral drift, fiducial markers can be used to characterize and correct for axial drift, although this requires the use of an astigmatic lens and a feedback mechanism to correct the sample position whilst the images are being acquired 43 . Commercial super-resolution systems will usually include some sort of stability control or focus-lock mechanism, which may be a more practical solution for correcting focus drift.…”

“…Drift is a well-recognized problem and there are a number of strategies that can be used to minimize it 19 or correct it post-acquisition either using fiducial markers 21,43 or cross-correlations 44 . In order to measure drift and correct for it, fluorescent beads of 100 nm diameter can be used as fiducial markers ( Figure 6D).…”