Microscope calibration using laser written fluorescence.

Alexander D Corbett,Patrick S Salter,Lothar Schermelleh,Andrew Yacoot,Martin Booth,Tony Wilson,Andrew Jefferson,Michael Shaw

doi:10.1364/oe.26.021887

Alexander D Corbett, Patrick S Salter + Show 6 more

Open Access

https://doi.org/10.1364/oe.26.021887

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Abstract

There is currently no widely adopted standard for the optical characterization of fluorescence microscopes. We used laser written fluorescence to generate two- and three-dimensional patterns to deliver a quick and quantitative measure of imaging performance. We report on the use of two laser written patterns to measure the lateral resolution, illumination uniformity, lens distortion and color plane alignment using confocal and structured illumination fluorescence microscopes.

Highlights

Fluorescence microscopes are essential tools across a wide range of scientific research
Whilst standards have been proposed in coherent microscopy [2], there is no single calibration standard that has been widely adopted in fluorescence microscopy that is able to provide a measure of key imaging performance parameters
The array of 8 × 8 × 3 features was imaged on a laser scanning confocal microscope build around an Olympus IX81

Summary

Introduction

Fluorescence microscopes are essential tools across a wide range of scientific research. The greater repeatability of sample preparation and specificity of fluorescent labels have led to an increased emphasis on extracting quantitative data from images rather than making qualitative observations [1]. Fluorescent beads with diameters smaller than the diffraction limit, provide a popular means of measuring the point spread function (PSF) across the field of view. A further limitation is that the subdiffraction size of the bead often results in (i) a weak fluorescence signal with low signal-tobackground ratio and (ii) only a few sampling points across the PSF. Both of these factors contribute to a high degree of error in the PSF measurement. As the beads themselves are distributed randomly across the field of view, it is not possible to extract any measures of image distortion across the field of view

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Conclusion