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Enabling Hours-Long Drift Correction with Nanometer Resolution in Optical Microscopy through Reflection
Binh Phan1, Michael R Stoneman1, Sabita Sharma1
1Department of Physics, University of Wisconsin-Milwaukee, 3135 N. Maryland Ave., Milwaukee, Wisconsin 53211, United States.
Nano Letters
|February 4, 2025
Summary
Focus readjustment for enhanced vertical resolution (FREVR) uses fiducial beads to precisely track focus, enabling hours of stable microscopy. This technique significantly enhances long-term imaging of biological samples.
Area of Science:
- Biophysics
- Optical Microscopy
- Cell Biology
Background:
- Optical microscopy is vital for real-time biological dynamics.
- Focal drift limits microscopy duration to minutes without mitigation.
- Long-term imaging requires precise focus stabilization.
Purpose of the Study:
- Introduce focus readjustment for enhanced vertical resolution (FREVR).
- Develop a method to overcome focal drift in microscopy.
- Enhance temporal and spatial resolution in long-term microscopy.
Main Methods:
- Utilize micrometer-sized fiducial beads alongside specimens.
- Track interference fringes from beads for nanometer-precision focus detection.
- Employ high-speed CMOS and sensitive EMCCD cameras for bead tracking and fluorescence quantification.
Main Results:
- Achieved stable, in-focus measurements for several hours.
- Demonstrated efficacy on single-molecule systems and mammalian cells.
- Successfully imaged labeled actin filaments and membrane proteins.
Conclusions:
- FREVR significantly extends microscopy stability and duration.
- The method offers nanometer-level focus precision.
- FREVR has broad potential to advance microscopy techniques.
