Soft Condensed Matter and Molecular Biophysics, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, the Netherlands.
Department of Imaging Physics, Delft University of Technology, Delft, the Netherlands.
Commun Biol. 2021 Jul 23;4(1):909. doi: 10.1038/s42003-021-02432-3.
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.
需要使用多个样本来监测和优化受激发射损耗（STED）和共聚焦显微镜的定量测量的质量和可靠性。在这里，我们提出了一个单一的样本来校准这些显微镜，调整它们的激光束，并在 3D 中测量它们的点扩散函数（PSF）。该样本由具有荧光和金核的折射率匹配的二氧化硅珠胶体晶体组成。显微镜可以使用晶体的周期性在三维空间中进行校准；可以使用金核的反射检查激光束的对准情况；可以使用荧光核在多个位置和深度测量 PSF。本文展示了如何使用该样本可视化和改善 STED 和共聚焦显微镜图像的质量。该样本可进行调整以满足不同数值孔径物镜和显微镜技术的要求，此外还可以用于定量评估作为深度函数的折射率失配。
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