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