3D test sample for the calibration and quality control of super-resolution and confocal microscopes

Abstract

A multitude of samples is required to monitor and optimize the quality and reliability of quantitative measurements of (super-resolution) light microscopes. Here, we present a single sample to calibrate 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 microscope 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 confocal and super-resolution 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.

Electron-beam patterned calibration structures for structured illumination microscopy

“…Current schemes for generating commercial light microscopy calibration standards rely on one- or two-photon optical lithography 13 , which does not reach the desired sub-diffraction limited resolution. Point spread function evaluation can be done using individual precisely defined, highly monodisperse fluorescent nanobeads or refractive-index matched colloidal crystal structures 14 with randomly dispersed fluorescent beads. Evaluation of the full, multi-scale modulation transfer function (MTF) however still poses challenges, e.g.…”

Synthesis, microscopy and manipulation of colloidal matter

“…This was equipped with a Leica h c p l a p o 93×/1.30 glyc motcorr s t e d white objective with a correction collar and an 85 w% glycerol/water mixture (n 21 D = 1.4506, V 21 D = 47.1) as immersion. The axial focal shift due to the small r i mismatch of this immersion with the sample medium should be only ∼ 2% [160], so the stage displacements are quoted as the z heights in this chapter. For excitation, a 543 nm laser line (80 MHz pulsed white light laser, n k t Photonics) was used and a 660 nm continuous wave laser provided the depletion light in s t e d measurements.…”