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1.
Methods Cell Biol ; 187: 117-137, 2024.
Article En | MEDLINE | ID: mdl-38705622

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.


Lung , Vimentin , Humans , Vimentin/metabolism , Phosphorylation , Lung/metabolism , Lung/ultrastructure , Microscopy, Fluorescence/methods , Pulmonary Artery/metabolism , Pulmonary Artery/cytology , Pulmonary Artery/ultrastructure , Fluorescent Antibody Technique/methods , Endothelial Cells/metabolism , Endothelial Cells/ultrastructure , Microscopy, Electron, Transmission/methods , Microscopy, Electron/methods
2.
FEBS Lett ; 598(10): 1127-1142, 2024 May.
Article En | MEDLINE | ID: mdl-38726814

Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near-atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD-related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology.


Lipid Droplets , Microscopy, Electron , Lipid Droplets/metabolism , Lipid Droplets/ultrastructure , Lipid Droplets/chemistry , Humans , Animals , Microscopy, Electron/methods , Lipid Metabolism
3.
Nutrients ; 16(9)2024 May 04.
Article En | MEDLINE | ID: mdl-38732636

(1) Background: parenteral nutrition (PN) is indispensable for patients unable to receive oral or enteral feeding. However, the complexity of PN solutions presents challenges regarding stability and compatibility. Precipitation reactions may occur. The most frequent is the formation of calcium phosphate (Ca-P). The different factors influencing these reactions must be considered to ensure patient safety. (2) Methods: eight paediatric PN solutions were prepared, following standard protocols. Samples were stored at room temperature and in a refrigerator. Electron microscopy, coupled with energy dispersive X-ray spectroscopy (EDS), was employed. Precipitates were analysed for composition and morphology. (3) Results: precipitates were observed in all samples, even at day 0. Crystalline structures, predominantly composed of calcium or magnesium, sometimes associated with chlorine or phosphorus, were detected. Additionally, amorphous precipitates, contained heterogeneous compositions, including unexpected elements, were identified. (4) Conclusions: various precipitates, primarily calcium- or magnesium-based, can form in PN solutions, although it is not expected that they can form under the real conditions of use. Calcium oxalate precipitation has been characterised, but the use of organic calcium and phosphate salts appears to mitigate calcium phosphate precipitation. Electron microscopy provides interesting results on NP precipitation, but sample preparation may present technical limitations that affect the interpretation of the results.


Calcium Phosphates , Chemical Precipitation , Drug Stability , Parenteral Nutrition Solutions , Parenteral Nutrition Solutions/chemistry , Calcium Phosphates/chemistry , Humans , Parenteral Nutrition , Spectrometry, X-Ray Emission , Microscopy, Electron , Magnesium/chemistry , Calcium/chemistry , Calcium/analysis
4.
Methods Cell Biol ; 187: 1-41, 2024.
Article En | MEDLINE | ID: mdl-38705621

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.


Microscopy, Electron, Transmission , Humans , Microscopy, Electron, Transmission/methods , Animals , Cryoelectron Microscopy/methods , Microscopy, Electron/methods , Microscopy, Immunoelectron/methods , Microscopy, Fluorescence/methods , Freeze Substitution/methods
5.
Methods Cell Biol ; 187: 73-97, 2024.
Article En | MEDLINE | ID: mdl-38705631

Cells are dynamic machines that continuously change their architecture to adapt and respond to extracellular and intracellular stimuli. Deciphering dynamic processes with nanometer-scale resolution inside cells is critical for mechanistic understanding. Here, we present a protocol that enables the in situ study of dynamic changes in intracellular structures under close-to-native conditions at high spatiotemporal resolution. Importantly, the cells are grown, transported, and imaged in a chamber in which environmental conditions such as temperature and gas (e.g., carbon dioxide or oxygen) concentration can be controlled. We demonstrate this protocol to quantify ultrastructural changes that occur during the cell cycle of cultured mammalian cells. The environment control system opens up the possibility of applying this method to primary cells, tissues, and organoids by adjusting environmental conditions.


Cell Cycle , Humans , Animals , Microscopy, Electron/methods
6.
Methods Cell Biol ; 187: 99-116, 2024.
Article En | MEDLINE | ID: mdl-38705632

Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.


COVID-19 , SARS-CoV-2 , SARS-CoV-2/ultrastructure , Humans , COVID-19/virology , Vero Cells , Chlorocebus aethiops , Animals , Microscopy, Electron, Transmission/methods , Virus Replication , Microscopy, Electron/methods
7.
Methods Cell Biol ; 187: 43-56, 2024.
Article En | MEDLINE | ID: mdl-38705629

Correlative Light Electron Microscopy (CLEM) encompasses a wide range of experimental approaches with different degrees of complexity and technical challenges where the attributes of both light and electron microscopy are combined in a single experiment. Although the biological question always determines what technology is the most appropriate, we generally set out to apply the simplest workflow possible. For 2D cell cultures expressing fluorescently tagged molecules, we report on a simple and very powerful CLEM approach by using gridded finder imaging dishes. We first determine the gross localization of the fluorescence using light microscopy and subsequently we retrace the origin/localization of the fluorescence by projecting it onto the ultrastructural reference space obtained by transmission electron microscopy (TEM). Here we describe this workflow and highlight some basic principles of the sample preparation for such a simple CLEM experiment. We will specifically focus on the steps following the resin embedding for TEM and the introduction of the sample in the electron microscope.


Workflow , Humans , Microscopy, Fluorescence/methods , Microscopy, Electron, Transmission/methods , Microscopy, Electron/methods , Animals
8.
Methods Cell Biol ; 187: 57-72, 2024.
Article En | MEDLINE | ID: mdl-38705630

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.


Hippocampus , Receptors, AMPA , Synapses , Animals , Mice , Hippocampus/metabolism , Hippocampus/cytology , Receptors, AMPA/metabolism , Synapses/metabolism , Synapses/ultrastructure , Membrane Proteins/metabolism , Gold/chemistry , Microscopy, Electron/methods , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism
9.
Methods Mol Biol ; 2800: 89-102, 2024.
Article En | MEDLINE | ID: mdl-38709480

In recent years, Correlative Multimodal Imaging (CMI) has become an "en vogue" technique and a bit of a buzzword. It entails combining information from different imaging modalities to extract more information from a sample that would otherwise not be possible from each individual technique. The best established CMI technology is correlative light and electron microscopy (CLEM), which applies light and electron microscopy on the exact same sample/structure. In general, it entails the detection of fluorescently tagged proteins or structures by light microscopy and subsequently their relative intracellular localization is determined with nanometer resolution using transmission electron microscopy (TEM). Here, we describe the different steps involved in a "simple" CLEM approach. We describe the overall workflow, instrumentation, and basic principles of sample preparation for a CLEM experiment exploiting stable expression of fluorescent proteins.


Microscopy, Electron, Transmission , Humans , Microscopy, Electron, Transmission/methods , Microscopy, Fluorescence/methods , Microscopy, Electron/methods , Image Processing, Computer-Assisted/methods , Animals
10.
Cell ; 187(10): 2574-2594.e23, 2024 May 09.
Article En | MEDLINE | ID: mdl-38729112

High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.


Drosophila melanogaster , Microscopy, Electron , Neurotransmitter Agents , Synapses , Animals , Brain/ultrastructure , Brain/metabolism , Connectome , Drosophila melanogaster/ultrastructure , Drosophila melanogaster/metabolism , gamma-Aminobutyric Acid/metabolism , Microscopy, Electron/methods , Neural Networks, Computer , Neurons/metabolism , Neurons/ultrastructure , Neurotransmitter Agents/metabolism , Synapses/ultrastructure , Synapses/metabolism
11.
Methods Mol Biol ; 2807: 93-110, 2024.
Article En | MEDLINE | ID: mdl-38743223

Correlative light-electron microscopy (CLEM) has evolved in the last decades, especially after significant developments in sample preparation, imaging acquisition, software, spatial resolution, and equipment, including confocal, live-cell, super-resolution, and electron microscopy (scanning, transmission, focused ion beam, and cryo-electron microscopy). However, the recent evolution of different laser-related techniques, such as mass spectrometry imaging (MSI) and laser capture microdissection, could further expand spatial imaging capabilities into high-resolution OMIC approaches such as proteomic, lipidomics, small molecule, and drug discovery. Here, we will describe a protocol to integrate the detection of rare viral reservoirs with imaging mass spectrometry.


HIV Infections , Humans , HIV Infections/virology , HIV-1/physiology , Mass Spectrometry/methods , Microscopy, Electron/methods , Molecular Imaging/methods , Disease Reservoirs/virology
12.
J Plant Physiol ; 297: 154236, 2024 Jun.
Article En | MEDLINE | ID: mdl-38621330

Germline cells are critical for transmitting genetic information to subsequent generations in biological organisms. While their differentiation from somatic cells during embryonic development is well-documented in most animals, the regulatory mechanisms initiating plant germline cells are not well understood. To thoroughly investigate the complex morphological transformations of their ultrastructure over developmental time, nanoscale 3D reconstruction of entire plant tissues is necessary, achievable exclusively through electron microscopy imaging. This paper presents a full-process framework designed for reconstructing large-volume plant tissue from serial electron microscopy images. The framework ensures end-to-end direct output of reconstruction results, including topological networks and morphological analysis. The proposed 3D cell alignment, denoise, and instance segmentation pipeline (3DCADS) leverages deep learning to provide a cell instance segmentation workflow for electron microscopy image series, ensuring accurate and robust 3D cell reconstructions with high computational efficiency. The pipeline involves five stages: the registration of electron microscopy serial images; image enhancement and denoising; semantic segmentation using a Transformer-based neural network; instance segmentation through a supervoxel-based clustering algorithm; and an automated analysis and statistical assessment of the reconstruction results, with the mapping of topological connections. The 3DCADS model's precision was validated on a plant tissue ground-truth dataset, outperforming traditional baseline models and deep learning baselines in overall accuracy. The framework was applied to the reconstruction of early meiosis stages in the anthers of Arabidopsis thaliana, resulting in a topological connectivity network and analysis of morphological parameters and characteristics of cell distribution. The experiment underscores the 3DCADS model's potential for biological tissue identification and its significance in quantitative analysis of plant cell development, crucial for examining samples across different genetic phenotypes and mutations in plant development. Additionally, the paper discusses the regulatory mechanisms of Arabidopsis thaliana's germline cells and the development of stamen cells before meiosis, offering new insights into the transition from somatic to germline cell fate in plants.


Imaging, Three-Dimensional , Imaging, Three-Dimensional/methods , Microscopy, Electron/methods , Arabidopsis/ultrastructure , Arabidopsis/growth & development , Arabidopsis/cytology , Algorithms , Plant Cells/ultrastructure , Image Processing, Computer-Assisted/methods
13.
Nat Commun ; 15(1): 2965, 2024 Apr 05.
Article En | MEDLINE | ID: mdl-38580652

VGluT3-expressing mouse retinal amacrine cells (VG3s) respond to small-object motion and connect to multiple types of bipolar cells (inputs) and retinal ganglion cells (RGCs, outputs). Because these input and output connections are intermixed on the same dendrites, making sense of VG3 circuitry requires comparing the distribution of synapses across their arbors to the subcellular flow of signals. Here, we combine subcellular calcium imaging and electron microscopic connectomic reconstruction to analyze how VG3s integrate and transmit visual information. VG3s receive inputs from all nearby bipolar cell types but exhibit a strong preference for the fast type 3a bipolar cells. By comparing input distributions to VG3 dendrite responses, we show that VG3 dendrites have a short functional length constant that likely depends on inhibitory shunting. This model predicts that RGCs that extend dendrites into the middle layers of the inner plexiform encounter VG3 dendrites whose responses vary according to the local bipolar cell response type.


Amacrine Cells , Retina , Mice , Animals , Amacrine Cells/physiology , Retina/physiology , Retinal Ganglion Cells/physiology , Synapses/metabolism , Microscopy, Electron , Dendrites/physiology
14.
Biochemistry (Mosc) ; 89(2): 257-268, 2024 Feb.
Article En | MEDLINE | ID: mdl-38622094

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aß42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aß42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.


Electrons , Mitochondrial Diseases , Animals , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Microscopy, Electron , Mitochondrial Diseases/metabolism
15.
Nat Commun ; 15(1): 3424, 2024 Apr 23.
Article En | MEDLINE | ID: mdl-38654023

Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral ß3-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.


Anti-Bacterial Agents , Cell Membrane , Molecular Dynamics Simulation , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Cell Membrane/drug effects , Lipopolysaccharides/pharmacology , Microbial Sensitivity Tests , Peptides/chemistry , Peptides/pharmacology , Microscopy, Electron , Gram-Negative Bacteria/drug effects , Escherichia coli/drug effects
16.
Sci Adv ; 10(16): eadk0217, 2024 Apr 19.
Article En | MEDLINE | ID: mdl-38630809

Biological phenomena, from enzymatic catalysis to synaptic transmission, originate in the structural transformations of biomolecules and biomolecular assemblies in liquid water. However, directly imaging these nanoscopic dynamics without probes or labels has been a fundamental methodological challenge. Here, we developed an approach for "electron videography"-combining liquid phase electron microscopy with molecular modeling-with which we filmed the nanoscale structural fluctuations of individual, suspended, and unlabeled membrane protein nanodiscs in liquid. Systematic comparisons with biochemical data and simulation indicate the graphene encapsulation involved can afford sufficiently reduced effects of the illuminating electron beam for these observations to yield quantitative fingerprints of nanoscale lipid-protein interactions. Our results suggest that lipid-protein interactions delineate dynamically modified membrane domains across unexpectedly long ranges. Moreover, they contribute to the molecular mechanics of the nanodisc as a whole in a manner specific to the protein within. Overall, this work illustrates an experimental approach to film, quantify, and understand biomolecular dynamics at the nanometer scale.


Electrons , Nanostructures , Membrane Proteins/chemistry , Molecular Dynamics Simulation , Microscopy, Electron , Lipids/chemistry , Lipid Bilayers/chemistry , Nanostructures/chemistry
17.
Eur J Histochem ; 68(1)2024 Feb 23.
Article En | MEDLINE | ID: mdl-38568205

The Feulgen reaction has been the first specific method for detecting DNA available at light microscopy since 1924. However, a similar specific method was proposed for electron microscopy only 50 years later. Here, we discuss the problems encountered in finding the electrondense reagent capable of taking advantage of the extremely high resolution offered by electron microscopy as well as some applications of the method.


Coloring Agents , Microscopy, Electron
18.
J Nanobiotechnology ; 22(1): 106, 2024 Mar 11.
Article En | MEDLINE | ID: mdl-38468300

Understanding the intricate nanoscale architecture of neuronal myelin during central nervous system development is of utmost importance. However, current visualization methods heavily rely on electron microscopy or indirect fluorescent method, lacking direct and real-time imaging capabilities. Here, we introduce a breakthrough near-infrared emissive curcumin-BODIPY derivative (MyL-1) that enables direct visualization of myelin structure in brain tissues. The remarkable compatibility of MyL-1 with stimulated emission depletion nanoscopy allows for unprecedented super-resolution imaging of myelin ultrastructure. Through this innovative approach, we comprehensively characterize the nanoscale myelinogenesis in three dimensions over the course of brain development, spanning from infancy to adulthood in mouse models. Moreover, we investigate the correlation between myelin substances and Myelin Basic Protein (MBP), shedding light on the essential role of MBP in facilitating myelinogenesis during vertebral development. This novel material, MyL-1, opens up new avenues for studying and understanding the intricate process of myelinogenesis in a direct and non-invasive manner, paving the way for further advancements in the field of nanoscale neuroimaging.


Boron Compounds , Curcumin , Animals , Mice , Brain/diagnostic imaging , Brain/metabolism , Neurons , Microscopy, Electron
19.
Methods Mol Biol ; 2793: 163-174, 2024.
Article En | MEDLINE | ID: mdl-38526730

Electron microscopy (EM) techniques play a vital role in virology research including phage discovery and their identification. The use of different staining protocols based on the concept of negative staining is one of the most important steps in the EM processing. This chapter will summarize the widely used EM protocols in phage research, their advantages, and limitations. Phage-based therapy, especially recently developed nanoparticle-phage conjugates, are expected to find clinical significance in the antimicrobial resistance (AMR) epidemic. EM techniques are important to characterize these conjugates and we will also discuss the methods here.


Bacteriophages , Epidemics , Microscopy, Electron , Negative Staining , Staining and Labeling
20.
Commun Biol ; 7(1): 377, 2024 Mar 28.
Article En | MEDLINE | ID: mdl-38548849

Mitochondria are the main suppliers of energy for cells and their bioenergetic function is regulated by mitochondrial dynamics: the constant changes in mitochondria size, shape, and cristae morphology to secure cell homeostasis. Although changes in mitochondrial function are implicated in a wide range of diseases, our understanding is challenged by a lack of reliable ways to extract spatial features from the cristae, the detailed visualization of which requires electron microscopy (EM). Here, we present a semi-automatic method for the segmentation, 3D reconstruction, and shape analysis of mitochondria, cristae, and intracristal spaces based on 2D EM images of the murine hippocampus. We show that our method provides a more accurate characterization of mitochondrial ultrastructure in 3D than common 2D approaches and propose an operational index of mitochondria's internal organization. With an improved consistency of 3D shape analysis and a decrease in the workload needed for large-scale analysis, we speculate that this tool will help increase our understanding of mitochondrial dynamics in health and disease.


Mitochondrial Membranes , Volume Electron Microscopy , Mice , Animals , Mitochondrial Membranes/metabolism , Mitochondria/metabolism , Energy Metabolism , Microscopy, Electron
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