@article{pmid34302049,

title = {3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes},

author = {Ernest B van der Wee and Jantina Fokkema and Chris L Kennedy and Marc Del Pozo and D A Matthijs de Winter and Peter N A Speets and Hans C Gerritsen and Alfons van Blaaderen},

doi = {10.1038/s42003-021-02432-3},

issn = {2399-3642},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {Commun Biol},

volume = {4},

number = {1},

pages = {909},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{van_hest_towards_2019,

title = {Towards robust and versatile single nanoparticle fiducial markers for correlative light and electron microscopy},

author = { Jacobine J.H.A. van Hest and Alexandra V. Agronskaia and Jantina Fokkema and F. Montanarella and A. Gregorio Puig and Celso de Mello Donega and Andries Meijerink and Gerhard A. Blab and Hans C. Gerritsen},

doi = {10.1111/jmi.12778},

year  = {2019},

date = {2019-01-01},

journal = {Journal of Microscopy},

volume = {274},

number = {1},

pages = {13--22},

abstract = {Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye-labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold-core rhodamine B-labelled silica particles, 15 nm CdSe/CdS/ZnS core–shell–shell quantum dots (QDs) and 230 nm Y 2 O 3 :Eu 3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold-core rhodamine B-labelled silica NPs and QDs are quenched after a single exposure to 60 ke −  nm –2 with an energy of 120 keV, while Y 2 O 3 :Eu 3+ NPs are robust and still show luminescence after five doses of 60 ke − nm –2 . In addition, the luminescence intensity of Y 2 O 3 :Eu 3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y 2 O 3 :Eu 3+ NPs are promising as robust fiducial marker in CLEM. Lay Description: Luminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye-labelled silica particles, quantum dots and lanthanide-doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye-labelled silica NPs and QDs are quenched after a single exposure to 60 ke − nm –2 with an energy of 120 keV, while lanthanide-doped inorganic NPs are robust and still show luminescence after five doses of 60 ke − nm –2 . In addition, the luminescence intensity of lanthanide-doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide-doped NPs are promising as robust fiducial marker in CLEM. © 2019 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mohammadian_high_2019,

title = {High accuracy, fiducial marker-based image registration of correlative microscopy images},

author = {Sajjad Mohammadian and Jantina Fokkema and Alexandra V Agronskaia and N Liv and C de Heus and E van Donselaar and Gerhard A. Blab and J Klumperman and Hans C Gerritsen},

doi = {10.1038/s41598-019-40098-4},

year  = {2019},

date = {2019-01-01},

journal = {Scientific Reports},

volume = {9},

number = {1},

abstract = {Fluorescence microscopy (FM) and electron microscopy (EM) are complementary techniques. FM affords examination of large fields of view and identifying regions of interest but has a low resolution. EM exhibits excellent resolution over a limited field of view. The combination of these two techniques, correlative microscopy, received considerable interest in the past years and has proven its potential in biology and material science. Accurate correlation of FM and EM images is, however, challenging due to the differences in contrast mechanism, size of field of view and resolution. We report an accurate, fast and robust method to correlate FM and EM images using low densities of fiducial markers. Here, 120 nm diameter fiducial markers consisting of fluorescently labelled silica coated gold nanoparticles are used. The method relies on recording FM, low magnification EM and high magnification EM images. Two linear transformation matrices are constructed, FM to low magnification EM and low magnification EM to high magnification EM. Combination of these matrices results in a high accuracy transformation of FM to high magnification EM coordinates. The method was tested using two different transmission electron microscopes and different Tokuyasu and Lowicryl sections. The overall accuracy of the correlation method is high, 5–30 nm. © 2019, The Author(s).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fokkema2018,

title = {Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy},

author = {Jantina Fokkema and Job Fermie and Nalan Liv and Dave J. van den Heuvel and Tom O. M. Konings and Gerhard A. Blab and Andries Meijerink and Judith Klumperman and Hans C. Gerritsen},

url = {https://doi.org/10.1038/s41598-018-31836-1},

doi = {10.1038/s41598-018-31836-1},

year  = {2018},

date = {2018-09-11},

journal = {Scientific Reports},

volume = {8},

number = {1},

pages = {13625},

abstract = {In this work, gold nanoparticles coated with a fluorescently labelled (rhodamine B) silica shell are presented as fiducial markers for correlative light and electron microscopy (CLEM). The synthesis of the particles is optimized to obtain homogeneous, spherical core-shell particles of arbitrary size. Next, particles labelled with different fluorophore densities are characterized to determine under which conditions bright and (photo)stable particles can be obtained. 2 and 3D CLEM examples are presented where optimized particles are used for correlation. In the 2D example, fiducials are added to a cryosection of cells whereas in the 3D example cells are imaged after endocytosis of the fiducials. Both examples demonstrate that the particles are clearly visible in both modalities and can be used for correlation. Additionally, the recognizable core-shell structure of the fiducials proves to be very powerful in electron microscopy: it makes it possible to irrefutably identify the particles and makes it easy to accurately determine the center of the fiducials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}