Combinatorial sample- and back-focal-plane imaging. Pt. I: Instrument and acquisition parameters affecting BFP images...

Area of Science:

• Optical microscopy
• Biophysics
• Fluorescence imaging

Background:

• The back-focal plane (BFP) contains rich information about fluorophores, including axial position, orientation, and local refractive index.
• BFP imaging is underutilized in biological fluorescence microscopy due to challenges with low-light and short-exposure conditions.
• Existing methods lack standardization and robustness for biological applications.

Purpose of the Study:

• To develop a reliable, real-time BFP imaging technique for biological fluorescence microscopy.
• To establish a framework for accurate axial fluorophore localization and refractive index measurements.
• To enable reproducible and scalable BFP imaging for dynamic biological systems.

Main Methods:

• Systematic analysis of key parameters affecting BFP image quality (Bertrand lens position, defocus, pixel size, binning).
• Development of hardware and software integration for multidimensional image series and online quality control.
• Evaluation of BFP imaging for supercritical-angle fluorescence (SAF) and undercritical-angle fluorescence (UAF) ratios.

Main Results:

• Demonstrated reliable, real-time BFP imaging under low-light and short-exposure conditions.
• Provided a robust framework for accurate axial fluorophore localization and near-membrane refractive index measurements.
• Achieved reduced experimental error and enhanced reproducibility through integrated hardware and software.

Conclusions:

• Transformed BFP imaging into a reproducible and scalable tool for surface-sensitive fluorescence microscopy.
• Laid the foundation for standardized BFP imaging protocols across laboratories.
• Opened avenues for machine-learning-based analysis pipelines in biological imaging.