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A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies

Ilya Olevsko, Kaitlin Szederkenyi, Jennifer Corridon, Aaron Au, Brigitte Delhomme, Thierry Bastien, Julien Fernandes, Christopher Yip, Martin Oheim, Adi Salomon
doi: https://doi.org/10.1101/2021.04.27.441588
Ilya Olevsko
1Institute of Nanotechnology and Advanced Materials (BINA), Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900 Israel
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Kaitlin Szederkenyi
2Université de Paris, CNRS, SPPIN - Saints-Pères Paris Institute for the Neurosciences, F-75006 Paris, France
3University of Toronto, Donnelly Centre for Cellular & Biomolecular Research, 160 College St, Toronto, ON M5S 3E1, Canada
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Jennifer Corridon
4Université de Paris, CNRS UMS 2009, INSERM US 36, BioMedTech Facilities, Campus Saint Germain, F-76006 Paris, France
5Université de Paris, Service Commun de Microscopie (SCM), Campus Saint Germain, F-76006 Paris, France
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Aaron Au
3University of Toronto, Donnelly Centre for Cellular & Biomolecular Research, 160 College St, Toronto, ON M5S 3E1, Canada
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Brigitte Delhomme
2Université de Paris, CNRS, SPPIN - Saints-Pères Paris Institute for the Neurosciences, F-75006 Paris, France
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Thierry Bastien
4Université de Paris, CNRS UMS 2009, INSERM US 36, BioMedTech Facilities, Campus Saint Germain, F-76006 Paris, France
6Université de Paris, Plateforme de Prototypage, Campus Saint Germain, F-76006 Paris, France
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Julien Fernandes
7UTechS Photonic BioImaging, C2RT, Institut Pasteur, F-75015 Paris, France
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Christopher Yip
3University of Toronto, Donnelly Centre for Cellular & Biomolecular Research, 160 College St, Toronto, ON M5S 3E1, Canada
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Martin Oheim
2Université de Paris, CNRS, SPPIN - Saints-Pères Paris Institute for the Neurosciences, F-75006 Paris, France
4Université de Paris, CNRS UMS 2009, INSERM US 36, BioMedTech Facilities, Campus Saint Germain, F-76006 Paris, France
5Université de Paris, Service Commun de Microscopie (SCM), Campus Saint Germain, F-76006 Paris, France
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  • For correspondence: adi.salomon@biu.ac.il martin.oheim@parisdescartes.fr
Adi Salomon
1Institute of Nanotechnology and Advanced Materials (BINA), Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900 Israel
2Université de Paris, CNRS, SPPIN - Saints-Pères Paris Institute for the Neurosciences, F-75006 Paris, France
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  • For correspondence: adi.salomon@biu.ac.il martin.oheim@parisdescartes.fr
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ABSTRACT

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

Graphical abstract Pyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective ×20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this “primitive” and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities.

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Research highlights

  • - highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies

  • - standard tissue paper presents features ranging from 400 nm to centimetres

  • - our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast

  • - it is easy to prepare, versatile, photostable and inexpensive

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵* Co-first authors

  • a This work was performed at the SPPIN (https://sppin.fr) during a sabbatical stay of AS during the academic year 2019-20. A Chateaubriand Senior Research Fellowship is gratefully acknowledged.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 27, 2021.
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A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies
Ilya Olevsko, Kaitlin Szederkenyi, Jennifer Corridon, Aaron Au, Brigitte Delhomme, Thierry Bastien, Julien Fernandes, Christopher Yip, Martin Oheim, Adi Salomon
bioRxiv 2021.04.27.441588; doi: https://doi.org/10.1101/2021.04.27.441588
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A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies
Ilya Olevsko, Kaitlin Szederkenyi, Jennifer Corridon, Aaron Au, Brigitte Delhomme, Thierry Bastien, Julien Fernandes, Christopher Yip, Martin Oheim, Adi Salomon
bioRxiv 2021.04.27.441588; doi: https://doi.org/10.1101/2021.04.27.441588

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