Combinatorial Sample-and Back-Focal-Plane (BFP) Imaging. Pt. I: Instrument and acquisition parameters affecting BFP images and their analysis
Journal:
arXiv
Published Date:
May 19, 2025
Abstract
The back-focal plane (BFP) of a high-numerical aperture objective contains
the fluoro-phore 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 (SAF) 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 SAF/UAF 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 multi-dimensional 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.
