Abstract
Multiple samples are required to monitor and optimize the quality and reliability of quantitative measurements of stimulated emission depletion (STED) and confocal microscopes. Here, we present a single sample to calibrate these microscopes, align their laser beams and measure their point spread function (PSF) in 3D. The sample is composed of a refractive index matched colloidal crystal of silica beads with fluorescent and gold cores. The microscopes can be calibrated in three dimensions using the periodicity of the crystal; the alignment of the laser beams can be checked using the reflection of the gold cores; and the PSF can be measured at multiple positions and depths using the fluorescent cores. It is demonstrated how this sample can be used to visualize and improve the quality of STED and confocal microscopy images. The sample is adjustable to meet the requirements of different NA objectives and microscopy techniques and additionally can be used to evaluate refractive index mismatches as a function of depth quantitatively.
| Original language | English |
|---|---|
| Article number | 909 |
| Pages (from-to) | 1-9 |
| Number of pages | 9 |
| Journal | Communications Biology |
| Volume | 4 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 23 Jul 2021 |
Bibliographical note
Funding Information:The authors are grateful to Dave van den Heuvel for the help with confocal measurements and particle synthesis, Peter Helfferich for the help with confocal (STED) and laser power measurements, Michiel Hermes for the particle identification in the FIB-SEM data and Job Fermie for the help with FIB-SEM sample preparation. This project was funded by the European Research Council under the European Union’s Seventh Framework Programme (FP-2007-2013)/ERC Advanced Grant Agreement 291667 HierarSACol, NWO - Microscopy Valley under project number 12715 and the Netherlands Center for Multiscale Catalytic Energy Conversion (MCEC), a NWO Gravitation programme funded by the Ministry of Education, Culture and Science of the government of the Netherlands.
Publisher Copyright:
© 2021, The Author(s).
Funding
The authors are grateful to Dave van den Heuvel for the help with confocal measurements and particle synthesis, Peter Helfferich for the help with confocal (STED) and laser power measurements, Michiel Hermes for the particle identification in the FIB-SEM data and Job Fermie for the help with FIB-SEM sample preparation. This project was funded by the European Research Council under the European Union’s Seventh Framework Programme (FP-2007-2013)/ERC Advanced Grant Agreement 291667 HierarSACol, NWO - Microscopy Valley under project number 12715 and the Netherlands Center for Multiscale Catalytic Energy Conversion (MCEC), a NWO Gravitation programme funded by the Ministry of Education, Culture and Science of the government of the Netherlands.