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

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell‐based organoids are promising as functional, self‐organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self‐organizing co‐culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration)

Bimodal endocytic probe for three-dimensional correlative light and electron microscopy

We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications

Bridging cellular dimensions by CLEM: novel tools for live-cell correlative imaging

Correlative live-cell light and electron microscopy (live-cell CLEM) has revolutionized bioimaging, since it is the only approach that infers molecular and dynamic information to ultrastructural context. CLEM is used to identify rare or transient events for EM analysis, that are nearly impossible to detect by electron microscopy (EM) alone. Live-cell fluorescence data can also be used to register live-cell dynamics to an ultrastructural snapshot. Finding back regions of interest (ROI) between modalities in 3D can prove challenging however. In addition, accurate overlay of fluorescence and EM data is crucial to interpret the correlated datasets. These challenges must be overcome to retrace individual organelles between live-cell FM data and EM. This thesis explored novel approaches to reliably facilitate live-cell correlative imaging. We demonstrate how correlative live-cell imaging and EM link dynamic and functional properties to ultrastructural context. Furthermore, we demonstrate how CLEM is used to significantly reduce imaging time in the EM by providing a system of reference points for targeting in focused ion beam scanning electron microscopy (FIB-SEM). Finally, we show the potential of different bimodal fiducial particles for (live-cell) CLEM using both 2D and 3D imaging systems. Overall, this thesis highlights the advances necessary to efficiently place live-cell dynamics and molecular composition into ultrastructural context, and demonstrates the benefits of the use of bimodal particles for more efficient CLEM applications. These, and other recent developments have made CLEM into an increasingly powerful tool in cell biology, by tightly interlinking the best that fluorescence and electron imaging have to offer

Bridging cellular dimensions by CLEM: novel tools for live-cell correlative imaging

Correlative live-cell light and electron microscopy (live-cell CLEM) has revolutionized bioimaging, since it is the only approach that infers molecular and dynamic information to ultrastructural context. CLEM is used to identify rare or transient events for EM analysis, that are nearly impossible to detect by electron microscopy (EM) alone. Live-cell fluorescence data can also be used to register live-cell dynamics to an ultrastructural snapshot. Finding back regions of interest (ROI) between modalities in 3D can prove challenging however. In addition, accurate overlay of fluorescence and EM data is crucial to interpret the correlated datasets. These challenges must be overcome to retrace individual organelles between live-cell FM data and EM. This thesis explored novel approaches to reliably facilitate live-cell correlative imaging. We demonstrate how correlative live-cell imaging and EM link dynamic and functional properties to ultrastructural context. Furthermore, we demonstrate how CLEM is used to significantly reduce imaging time in the EM by providing a system of reference points for targeting in focused ion beam scanning electron microscopy (FIB-SEM). Finally, we show the potential of different bimodal fiducial particles for (live-cell) CLEM using both 2D and 3D imaging systems. Overall, this thesis highlights the advances necessary to efficiently place live-cell dynamics and molecular composition into ultrastructural context, and demonstrates the benefits of the use of bimodal particles for more efficient CLEM applications. These, and other recent developments have made CLEM into an increasingly powerful tool in cell biology, by tightly interlinking the best that fluorescence and electron imaging have to offer

Functional characterization of endo-lysosomal compartments by correlative live-cell and volume electron microscopy

Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce "functional correlative microscopy" (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures

Functional characterization of endo-lysosomal compartments by correlative live-cell and volume electron microscopy

Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce “functional correlative microscopy” (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures

Granzymes A and K differentially potentiate LPS-induced cytokine response

Granzymes are serine proteases that, upon release from cytotoxic cells, induce apoptosis in tumor cells and virally infected cells. In addition, a role of granzymes in inflammation is emerging. Recently, we have demonstrated that extracellular granzyme K (GrK) potentiates lipopolysaccharide (LPS)-induced cytokine response from monocytes. GrK interacts with LPS, disaggregates LPS micelles, and stimulates LPS-CD14 binding and Toll-like receptor signaling. Here we show that human GrA also potentiates cytokine responses in human monocytes initiated by LPS or Gram-negative bacteria. Similar to GrK, this effect is independent of GrA catalytic activity. Unlike GrK, however, GrA does not bind to LPS, has little influence on LPS micelle disaggregation, and does not augment LPS-CD14 complex formation. We conclude that GrA and GrK differentially modulate LPS-Toll-like receptor signaling in monocytes, suggesting functional redundancy among cytotoxic lymphocyte proteases in the anti-bacterial innate immune response