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.
Fig: Calibration and alignment sample: a Transmission electron micrograph (TEM) of the two building blocks of the sample: monodisperse silica-coated 45 nm fluorescent silica (red) and 80 nm gold (black) cores with a total diameter of 505 nm. The bottom row depicts the fluorescent (left two) and gold core (right two) particles, as imaged with TEM (left) and as schematic (right) as used in (b). The cores of the schematic particles are not drawn to scale. Inset: upon (TEM) contrast variation the gold cores become visible. The scale bar is 500 nm. b Schematic of the sample as seen from the side consisting of a crystal composed of the particles in (a). c Top view of the sample showing the bright Bragg reflections of the index matched crystal upon illumination from the side (left) and full transparency under available light (right). The green marking on the sample was used to locate the crystal after refractive index matching. The direction of crystal growth during vertical deposition is parallel to the Y direction, as indicated by the white arrow in (c). The scale bars are 0.5 cm.
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.
van der Wee, E.B., Fokkema, J., Kennedy, C.L. et al. 3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes. Commun Biol 4, 909 (2021). https://doi.org/10.1038/s42003-021-02432-3