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

The back-focal plane (BFP) of a high-numerical aperture objective contains the fluorophore radiation pattern, which encodes information about the axial fluorophore position, molecular orientation and the local refractive index of the embedding medium. BFP image acquisition and analysis are common to conoscopy, k-space imaging, supercritical-angle fluorescence, and single-molecule detection, but they are rarely being used in biological fluorescence. This work addresses a critical gap in quantitative microscopy by enabling reliable, real-time BFP imaging under low-light conditions and/or short exposure times, typical of biological experiments. By systematically analyzing how key parameters-such as Bertrand lens position, defocus, pixel size, and binning-affect BFP image quality and supercritical-angle fluorescence/undercritical-angle fluorescence ratios, we provide a robust framework for accurate axial fluorophore localization and near-membrane refractive index measurements. The described hardware and software integration allows for multidimensional image series and online quality control, reducing experimental error and enhancing reproducibility. Our contributions lay the foundation for standardized BFP imaging across laboratories, expanding its application to dynamic biological systems, and opening the door to machine-learning-based analysis pipelines. Ultimately, this work transforms BFP imaging from an expert-dependent technique into a reproducible and scalable tool for surface-sensitive fluorescence microscopy.