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Quantitative high dynamic range beam proling for fluorescence microscopy.

Mitchell, T.J. and Saunter, C.D. and O'Nions, W. and Girkin, J.M. and Love, G.D. (2014) 'Quantitative high dynamic range beam proling for fluorescence microscopy.', Review of scientific instruments., 85 (10). p. 103713.


Modern developmental biology relies on optically-sectioning uorescence 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 (3-D) intensity prole of the illumination employed, the ability to directly record and analyze these proles is of great use to the uorescence microscopist or instrument builder. Though excitation beam proles can be measured indirectly using a sample of uorescent 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 proling device and high dynamic range ux reconstruction algorithm that together are capable of accurately reproducing quantitative 3-D ux maps over a large focal volume. Performance of this beam proling system is veried within an optical test bench and demonstrated for uorescence microscopy by proling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely- exible beam amplitude diagnostic tool for use within the life sciences.

Item Type:Article
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Publisher statement:© 2014 American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The following article appeared in Review of Scientific Instruments 85 (10), 103713 (2014) and may be found at
Date accepted:12 October 2014
Date deposited:03 November 2014
Date of first online publication:29 October 2014
Date first made open access:No date available

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