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Updated: Jul 20, 2026

Fluorescence Imaging with One-nanometer Accuracy (FIONA)
Published on: September 26, 2014
The sampling limit in fluorescence microscopy
Rainer Heintzmann1, Colin J R Sheppard
1King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London SE1 1UL, United Kingdom. Rainer.Heintzmann@kcl.ac.uk
Abstract:
Sampling in fluorescence microscopy is treated using the concept of the three-dimensional (3D) optical transfer function (OTF). The border of the OTF frequency surface defines the required minimum sampling. The shape of the OTF is derived from simple considerations and valid for far-field high numerical aperture, vector theory. Optimal regular sampling is achieved by a hexagonal grid in 2D, and corresponding hexagonal structures, body-centered cubic (bcc) and hexagonal close-packed (hcp) structures, in 3D. As compared to standard (rectilinear grid) sampling a reduction of 13.4% in 2D and 29.3% in 3D can be achieved with optimized sampling. This reduction in data size is also accompanied by an imaging speed improvement, a reduction of sample bleaching, and can lead to imaging with better signal to noise ratio.
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