Using fixed fiduciary markers for stage drift correction.

Area of Science:

• Microscopy and Nanotechnology
• Biophysics
• Instrumentation

Background:

• Super-resolution microscopy demands nanometric precision in sample stage stability.
• Existing methods for stage stabilization can be costly or computationally intensive.

Purpose of the Study:

• Introduce a cost-effective, low-computation method for nanometric stage stabilization.
• Enable precise tracking of sample movement during super-resolution imaging.

Main Methods:

• Utilize fiduciary markers (1 µm cubes) placed on coverslips for tracking.
• Employ Gaussian fitting to track x-y center of markers to 1 nm precision.
• Use out-of-focus light diffraction rings to track z-axis to < 5 nm (dry) or <17 nm (wet).
• Employ infrared light-emitting diodes (IR-LEDs) and a separate camera to avoid interference with visible fluorescence measurements.

Main Results:

• Demonstrated tracking of fiduciary markers with nanometric precision in x, y, and z.
• Successfully corrected for coverslip drift, enabling precise measurements.
• Applied the method to observe kinesin motor protein walking with approximately 8 nm steps.

Conclusions:

• The developed method offers a low-cost, accessible solution for achieving nanometric stage stability.
• This technique facilitates high-precision measurements and observations in super-resolution microscopy.
• The method is compatible with standard microscopy setups and does not interfere with fluorescence imaging.