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Department of Physics

Staff profile

Publication details for Professor Gordon Love

Mitchell, T.J., Saunter, C.D., O'Nions, W., Girkin, J.M. & Love, G.D. (2014). Quantitative High Dynamic Range Beam Pro ling for Fluorescence Microscopy. Review of Scientific Instruments 85(10): 103713.

Author(s) from Durham

Abstract

Modern developmental biology relies on optically sectioning fluorescence microscope techniques to
produce non-destructive in vivo images of developing specimens at high resolution in three dimensions.
As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity
profile of the illumination employed, the ability to directly record and analyze these profiles is of
great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles
can be measured indirectly using a sample of fluorescent beads and recording the emission along
the microscope detection path, we demonstrate an alternative approach where a miniature camera
sensor is used directly within the illumination beam. Measurements taken using our approach are
solely concerned with the illumination optics as the detection optics are not involved. We present a
miniature beam profiling device and high dynamic range flux reconstruction algorithm that together
are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance
of this beam profiling system is verified within an optical test bench and demonstrated for
fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination
microscope. The generality and success of this approach showcases a widely flexible beam amplitude
diagnostic tool for use within the life sciences.