3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes

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.