RT Journal Article
SR Electronic
T1 A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.04.27.441588
DO 10.1101/2021.04.27.441588
A1 Ilya Olevsko
A1 Kaitlin Szederkenyi
A1 Jennifer Corridon
A1 Aaron Au
A1 Brigitte Delhomme
A1 Thierry Bastien
A1 Julien Fernandes
A1 Christopher Yip
A1 Martin Oheim
A1 Adi Salomon
YR 2021
UL http://biorxiv.org/content/early/2021/04/27/2021.04.27.441588.abstract
AB 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.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 inexpensiveCompeting Interest StatementThe authors have declared no competing interest.