Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D

Bridging the Gap: 3D Real-Space Characterization of Colloidal Assemblies via FIB-SEM Tomography

Insight in the structure of nanoparticle assemblies up to a single particle level is key to understand the collective properties of these assemblies, which critically depend on the individual particle positions and orientations. However, the characterization of large, micron sized assemblies containing small, 10-500 nanometer, sized colloids is highly challenging and cannot easily be done with the conventional light, electron or X-ray microscopy techniques. Here, we demonstrate that focused ion beam-scanning electron microscopy (FIB-SEM) tomography in combination with image processing enables quantitative real-space studies of ordered and disordered particle assemblies too large for conventional transmission electron tomography, containing particles too small for confocal microscopy. First, we demonstrate the high resolution structural analysis of spherical nanoparticle assemblies, containing small anisotropic gold nanoparticles. Herein, FIB-SEM tomography allows the characterization of assembly dimensions which are inaccessible to conventional transmission electron microscopy. Next, we show that FIB-SEM tomography is capable of characterizing much larger ordered and disordered assemblies containing silica colloids with a diameter close to the resolution limit of confocal microscopes. We determined both the position and the orientation of each individual (nano)particle in the assemblies by using recently developed particle tracking routines. Such high precision structural information is essential in the understanding and design of the collective properties of new nanoparticle based materials and processes.Comment: 17 pages, 4 figures, Supplemental Information at articles webpage: https://doi.org/10.1039/C8NR09753

Gold/Silica Particles for Various Applications in (CLEM) Microscopy

The field of Correlative Light and Electron Microscopy (CLEM), has expanded rapidly in the last decade. Especially in biology it turns out to be very useful to combine these two techniques. Light microscopy, or more specifically fluorescence microscopy (FM), is used to visualize, localize, and track specific fluorescent molecules in cells over large areas with high sensitivity, while electron microscopy (EM) provides high resolution ultrastructural information of cells and materials. To gain additional information from CLEM experiments, it is important to properly overlay or register (stacks of) images obtained in the two modalities. This process is complicated by the vast different field of view of FM and EM, as well as the different contrast mechanisms of these techniques. Particles, or fiducial markers, that are visible in both modalities can be used to overcome this problem. In this work, nanocomposite core-shell particles based on a gold core and a fluorescently labelled silica shell are deployed as fiducial markers for 2D and 3D (integrated)CLEM. Within this particle, the gold core provides contrast for EM while fluorophores covalently incorporated in the silica shell provide fluorescence for FM. For FM, it is important that the fiducial markers are bright and visible over a longer period of time. As both properties are strongly influenced by the fluorophore labelling density this topic is also addressed. The work finishes with the presentation of a single sample based on similar core-shell particles to check and optimize the full performance of a high-end 3D super-resolution (STED) light microscope

Gold/Silica Particles for Various Applications in (CLEM) Microscopy

The field of Correlative Light and Electron Microscopy (CLEM), has expanded rapidly in the last decade. Especially in biology it turns out to be very useful to combine these two techniques. Light microscopy, or more specifically fluorescence microscopy (FM), is used to visualize, localize, and track specific fluorescent molecules in cells over large areas with high sensitivity, while electron microscopy (EM) provides high resolution ultrastructural information of cells and materials. To gain additional information from CLEM experiments, it is important to properly overlay or register (stacks of) images obtained in the two modalities. This process is complicated by the vast different field of view of FM and EM, as well as the different contrast mechanisms of these techniques. Particles, or fiducial markers, that are visible in both modalities can be used to overcome this problem. In this work, nanocomposite core-shell particles based on a gold core and a fluorescently labelled silica shell are deployed as fiducial markers for 2D and 3D (integrated)CLEM. Within this particle, the gold core provides contrast for EM while fluorophores covalently incorporated in the silica shell provide fluorescence for FM. For FM, it is important that the fiducial markers are bright and visible over a longer period of time. As both properties are strongly influenced by the fluorophore labelling density this topic is also addressed. The work finishes with the presentation of a single sample based on similar core-shell particles to check and optimize the full performance of a high-end 3D super-resolution (STED) light microscope

High accuracy, fiducial marker-based image registration of correlative microscopy images

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

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy

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

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy

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

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 quantitativel