Correlative Light Electron Microscopy Research Articles

Supramolecular Assembly in Live Cells Mapped by Real-Time Phasor-Fluorescence Lifetime Imaging.

The complex dynamics and transience of assembly pathways in living systems complicate the understanding of these molecular to nanoscale processes. Current technologies are unable to track the molecular events leading to the onset of assembly, where real-time information is imperative to correlate their rich biology. Using a chemically designed pro-assembling molecule, we map its transformation into nanofibers and their fusion with endosomes to form hollow fiber clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy and tomography (CLEM), spatiotemporal splicing of the assembly events shows time-correlated metabolic dysfunction. The biological impact begins with assembly-induced endosomal disruption that reduces glucose transport into the cells, which, in turn, stymies mitochondrial respiration.

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites

BackgroundThe infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria.ResultsThe liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples’ features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria.ConclusionsA CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.Graphical

Sensing the Spin State of Room-Temperature Switchable Cyanometallate Frameworks with Nitrogen-Vacancy Centers in Nanodiamonds.

Room-temperature magnetically switchable materials play a vital role in current and upcoming quantum technologies, such as spintronics, molecular switches, and data storage devices. The increasing miniaturization of device architectures produces a need to develop analytical tools capable of precisely probing spin information at the single-particle level. In this work, we demonstrate a methodology using negatively charged nitrogen vacancies (NV-) in fluorescent nanodiamond (FND) particles to probe the magnetic switching of a spin crossover (SCO) metal-organic framework (MOF), [Fe(1,6-naphthyridine)2(Ag(CN)2)2] material (1), and a single-molecule photomagnet [X(18-crown-6)(H2O)3]Fe(CN)6·2H2O, where X = Eu and Dy (materials 2a and 2b, respectively), in response to heat, light, and electron beam exposure. We employ correlative light-electron microscopy using transmission electron microscopy (TEM) finder grids to accurately image and sense spin-spin interacting particles down to the single-particle level. We used surface-sensitive optically detected magnetic resonance (ODMR) and magnetic modulation (MM) of FND photoluminescence (PL) to sense spins to a distance of ca. 10-30 nm. We show that ODMR and MM sensing was not sensitive to the temperature-induced SCO of FeII in 1 as formation of paramagnetic FeIII through surface oxidation (detected by X-ray photoelectron spectroscopy) on heating obscured the signal of bulk SCO switching. We found that proximal FNDs could effectively sense the chemical transformations induced by the 200 keV electron beam in 1, namely, AgI → Ag0 and FeII → FeIII. However, transformations induced by the electron beam are irreversible as they substantially disrupt the structure of MOF particles. Finally, we demonstrate NV- sensing of reversible photomagnetic switching, FeIII + (18-crown-6) ⇆ FeII + (18-crown-6)+ •, triggered in 2a and 2b by 405 nm light. The photoredox process of 2a and 2b proved to be the best candidate for room-temperature single-particle magnetic switching utilizing FNDs as a sensor, which could have applications into next-generation quantum technologies.

Giant Quantum Electrodynamic Effects on Single SiV Color Centers in Nanosized Diamonds.

Understanding and mastering quantum electrodynamics phenomena is essential to the development of quantum nanophotonics applications. While tailoring of the local vacuum field has been widely used to tune the luminescence rate and directionality of a quantum emitter, its impact on their transition energies is barely investigated and exploited. Fluorescent defects in nanosized diamonds constitute an attractive nanophotonic platform to investigate the Lamb shift of an emitter embedded in a dielectric nanostructure with high refractive index. Using spectral and time-resolved optical spectroscopy of single SiV defects, we unveil blue shifts (up to 80 meV) of their emission lines, which are interpreted from model calculations as giant Lamb shifts. Moreover, evidence for a positive correlation between their fluorescence decay rates and emission line widths is observed, as a signature of modifications not only of the photonic local density of states but also of the phononic one, as the nanodiamond size is decreased. Correlative light-electron microscopy of single SiVs and their host nanodiamonds further supports these findings. These results make nanodiamond-SiVs promising as optically driven spin qubits and quantum light sources tunable through nanoscale tailoring of vacuum-field fluctuations.

A novel optimized pre-embedding antibody-labelling correlative light electron microscopy technique.

In the intricate environment of a cell, many studies seek to discover the location of specific events or objects of interest. Advances in microscopy in recent years have allowed for high detail views of specific areas of cells of interest using correlative light electron microscopy (CLEM). While this powerful technique allows for the correlation of a specific area of fluorescence on a confocal microscope with that same area in an electron microscope, it is most often used to study tagged proteins of interest. This method adapts the correlative method for use with antibody labelling. We have shown that some cellular structures are more sensitive than others to this process and that this can be a useful technique for laboratories where tagged proteins or viruses, or dedicated CLEM instruments are not available.

Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.

Some tips and tricks for a Correlative Light Electron Microscopy workflow using stable expression of fluorescent proteins.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) culture and sample preparation for correlative light electron microscopy.

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.

Some Guiding Principles for a "Simple" Correlative Light Electron Microscopy Experiment.

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.

Correlative Imaging to Detect Rare HIV Reservoirs and Associated Damage in Tissues.

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.

Reduced ER-mitochondrial contact sites and mitochondrial Ca2+ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines.

Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca2+ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.

From Organ to Organelle: towards a Multimodal 3D Cell Atlas of the Malaria Parasite Mosquito stages using Correlative Light, X-ray and volume Electron Microscopy.

From Organ to Organelle: towards a Multimodal 3D Cell Atlas of the Malaria Parasite Mosquito stages using Correlative Light, X-ray and volume Electron Microscopy.

A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells.

A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells.

Astrocytic engagement of the corticostriatal synaptic cleft is disrupted in a mouse model of Huntington’s disease

Astroglial dysfunction contributes to the pathogenesis of Huntington's disease (HD), and glial replacement can ameliorate the disease course. To establish the topographic relationship of diseased astrocytes to medium spiny neuron (MSN) synapses in HD, we used 2-photon imaging to map the relationship of turboRFP-tagged striatal astrocytes and rabies-traced, EGFP-tagged coupled neuronal pairs in R6/2 HD and wild-type (WT) mice. The tagged, prospectively identified corticostriatal synapses were then studied by correlated light electron microscopy followed by serial block-face scanning EM, allowing nanometer-scale assessment of synaptic structure in 3D. By this means, we compared the astrocytic engagement of single striatal synapses in HD and WT brains. R6/2 HD astrocytes exhibited constricted domains, with significantly less coverage of mature dendritic spines than WT astrocytes, despite enhanced engagement of immature, thin spines. These data suggest that disease-dependent changes in the astroglial engagement and sequestration of MSN synapses enable the high synaptic and extrasynaptic levels of glutamate and K+ that underlie striatal hyperexcitability in HD. As such, these data suggest that astrocytic structural pathology may causally contribute to the synaptic dysfunction and disease phenotype of those neurodegenerative disorders characterized by network overexcitation.

Correlative light-electron microscopy: integrating dynamics to structure

A challenge of classical electron microscopy (EM) modalities is the static and limited view they present of dynamic biological processes. Correlative light and EM (CLEM) pairs the precise imaging of the cellular location in light microscopy (LM) and EM, taking advantage of both methods. Recent advances in protein labeling, fluorescent markers, super-resolution microscopy, and EM technologies have allowed widespread application in modern biological research. Because of the wide versatility of CLEM, it is difficult to describe a single workflow. In general, samples are prepared and imaged by LM before EM preparation and imaging, with software utilized for the correlation of the two images. Fluorescent tags can help to select and localize structures for EM or add dynamics through live-cell imaging. Future developments in multimodal probes showing electron density and fluorescence activity will allow greater integration of the modalities and high-throughput workflows. Can be paired with focused ion beam-scanning EM (FIB-SEM), cryo-EM, transmission EM (TEM), and serial block-face SEM (SBF-SEM), as well as structured illumination microscopy (SIM), stimulated emission depletion (STED), and total internal reflection fluorescence (TIRF). Amenable to live-cell imaging, in part due to advances that can quickly freeze a sample for cryo-EM. Systems to pair with microscopic X-ray computed tomography to provide non-destructive forms of 3D imaging alongside high-resolution micrographs obtained by TEM and fluorescence. Sensitive enough for applications such as virus–host cell membrane interactions. Alongside other techniques, can localize the distribution of specific proteins or structures within a sample and correlate this information with the ultrastructural details provided by EM. Finding the appropriate FM and EM microscopes may be difficult and expensive. Preparing samples for both modalities requires careful planning and optimization so they are properly preserved, labeled, and processed for the chosen application. The growth of cells on the grid during the LM phase can make correlational positioning difficult. Aligning LM and EM micrographs can be challenging and time-consuming due to differences in resolution and sample shrinkage. Lower throughput and smaller region-of-interest size than traditional FM techniques due to being reliant on EM acquisition and additional sample preparation times. We thank Kit Neikirk for his thoughtful suggestions and edits to the manuscript. Funding by the UNCF/Bristol-Myers Squibb E.E. Just Faculty Fund, BWF Career Awards at the Scientific Interface Award, BWF Ad-hoc Award, National Institutes of Health (NIH) Small Research Pilot Subaward to 5R25HL106365-12 from the NIH PRIDE Program, DK020593, Vanderbilt Diabetes and Research Training Center for DRTC Alzheimer’s Disease Pilot & Feasibility Program. CZI Science Diversity Leadership grant number 2022- 253529 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (to A.H.). The images were arranged using BioRender and all representative images are original. The authors have no interests to declare.

Extensive programmed centriole elimination unveiled in C. elegans embryos.

Centrioles are critical for fundamental cellular processes, including signaling, motility, and division. The extent to which centrioles are present after cell cycle exit in a developing organism is not known. The stereotypical lineage of Caenorhabditis elegans makes it uniquely well-suited to investigate this question. Using notably lattice light-sheet microscopy, correlative light electron microscopy, and lineage assignment, we found that ~88% of cells lose centrioles during embryogenesis. Our analysis reveals that centriole elimination is stereotyped, occurring invariably at a given time in a given cell type. Moreover, we established that experimentally altering cell fate results in corresponding changes in centriole fate. Overall, we uncovered the existence of an extensive centriole elimination program, which we anticipate to be paradigmatic for a broad understanding of centriole fate regulation.

An Iridium (III) complex revealing cytoskeleton nanostructures under super-resolution nanoscopy and liquid-phase electron microscopy

Live cell actin visualization is fundamental for exploring cellular motility, cytokinesis, intracellular transport, and other correlated functions. The current imaging techniques that allow imaging of actin in its native environment are optical and electron microscopy. Such imaging techniques offer high enough resolution to investigate the ultrastructure of actin however they come at the expense of actin integrity. Inspired by the lack of suitable probes that preserve actin's integrity, we designed a cyclometalated Ir (III) complex that interacts with live cells and displays light switch behaviour upon specific actin binding. The exceptional photophysical properties of the proposed probe allow unprecedented resolution of cytoskeleton ultrastructures under stimulated emission depletion (STED) super-resolution nanoscopy. Moreover, the Ir complex enables the capability of visualizing actin polymers and periodicity under correlative light electron microscopy (CLEM) and liquid-phase electron microscopy (LPEM) at ∼ 8 nm resolution.

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy.

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view. In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification. In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Correlative light-electron microscopy using small gold nanoparticles as single probes

Correlative light-electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 µm size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods.

Labelling strategies for correlative light electron microscopy.

Imaging is one of the key technologies underpinning discoveries in biomedical research. Each imaging technique however usually only provides a specific type of information. For instance, live-cell imaging using fluorescent tags can show us the dynamics of a system. On the other hand, electron microscopy (EM) gives us better resolution combined with the structural reference space. By applying a combination of light and electron microscopy modalities to a single sample one can exploit the advantages of both techniques in correlative light electron microscopy (CLEM). Although CLEM approaches can generate additional insights into the sample that cannot be gained by either technique in isolation, the visualization of the object of interest via markers or probes is still one of the bottlenecks in a Correlative Microscopy workflow. Whereas fluorescence is not directly visible in a standard electron microscope, gold particles, as the most common choice of probe for EM can also only be visualized using specialized light microscopes. In this review we will discuss some of the latest developments of probes for CLEM and some strategies how to choose a probe, discussing pros and cons of specific probes, and ensuring that they function as a dual modality marker.