Image-based 3D active sample stabilization on the nanometer scale for optical microscopy.

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Super-resolution microscopy requires long acquisition times, making it susceptible to sample drift.
• While lateral drift can be corrected post-acquisition, 3D drift correction during acquisition is challenging but crucial for data quality.
• Existing 3D active stabilization schemes can be complex and demanding.

Purpose of the Study:

• To present a straightforward active drift correction scheme for nanometer-scale 3D sample stabilization.
• To achieve high-precision 3D stabilization compatible with state-of-the-art super-resolution microscopy.
• To offer an accessible solution for enhancing imaging data collection.

Main Methods:

• Developed a refined algorithm for active drift correction adaptable to various reference structures.
• Integrated a simple widefield imaging path for 3D stabilization.
• Utilized an open-source control software with a graphical user interface for ease of implementation.

Main Results:

• Achieved ~1 nm 3D sample position stability using both high and low numerical aperture objective lenses.
• Demonstrated the scheme's effectiveness with diverse reference structures, not requiring sparse signal peaks.
• Validated the system's performance in enhancing data collection for single-molecule localization microscopy and cryo-confocal imaging.

Conclusions:

• The presented active drift correction scheme offers nanometer-scale 3D stability with improved implementation simplicity.
• This technology enhances data acquisition for demanding super-resolution and diffraction-limited imaging techniques.
• The open-source software facilitates widespread adoption and advancement in high-resolution microscopy.