Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.     

High-pressure freezing, cellular tomography, and structural cell biology

Structural cell biology, which we define as electron microscopic analysis of intact cells, suffered a loss of interest and activity following the advances in light microscopy beginning in the 1990s. Interestingly, it is the wealth of detailed observation in the light microscope that is one of the driving forces for the current renewed interest in electron microscopy (EM). A great many cellular details are simply beyond the resolving power of the light microscope. In this article, we describe how electron microscopists are responding to the demands for better preservation of cells and for ways to view cell ultrastructure in three dimensions at high resolution. We discuss how low temperature methods, especially high-pressure freezing and freeze substitution, reduce the artifacts of conventional EM specimen preparation. We also give a brief introduction to cellular electron tomography, a powerful analytical method that can give near-atomic resolution of cell ultrastructure in three-dimensional (3-D) models.     

Integrated Electron Microscopy: Super-Duper Resolution

Since its inception, electron microscopy (EM) has revealed that cellular membranes are organized into structurally distinct subdomains, created by localized protein and lipid assemblies to perform specific complex cellular functions. Caveolae are membrane subdomains that function as signaling platforms, endocytic carriers, sensors of membrane tension, and mechanical stress, as well as in lipid homeostasis. They were first discovered almost 60 years ago by pioneering electron microscopists. While new and exciting developments in SUPER-resolution fluorescent light microscopy facilitate studies of the spatial organization of fluorescently labeled protein components, these techniques cannot reveal the underlying cellular structures. Thus, equally exciting are developments in EM: genetically encoded probes for protein localization at sub-10 nm resolution, more powerful instruments that allow imaging of larger cell volumes, and computational methods for reconstructing three-dimensional images. Used in combination, as done by Ludwig et al. in the current issue of PLOS Biology, these tools reveal high-resolution insights into the composition and organization of the caveolae coat and the formation of these specialized structures. Together, these advances are contributing to a resurgence in EM.     

Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy

Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques.     

Lamin B distribution and association with peripheral chromatin revealed by optical sectioning and electron microscopy tomography

We have used a combination of immunogold staining, optical sectioning light microscopy, intermediate voltage electron microscopy, and EM tomography to examine the distribution of lamin B over the nuclear envelope of CHO cells. Apparent inconsistencies between previously published light and electron microscopy studies of nuclear lamin staining were resolved. At light microscopy resolution, an apparent open fibrillar network is visualized. Colocalization of lamin B and nuclear pores demonstrates that these apparent fibrils, separated by roughly 0.5 micron, are anti-correlated with the surface distribution of nuclear pores; pore clusters lie between or adjacent to regions of heavy lamin B staining. Examination at higher, EM resolution reveals that this apparent lamin B network does not correspond to an actual network of widely spaced, discrete bundles of lamin filaments. Rather it reflects a quantitative variation in lamin staining over a roughly 0.5-micron size scale, superimposed on a more continuous but still complex distribution of lamin filaments, spatially heterogeneous on a 0.1-0.2-micron size scale. Interestingly, lamin B staining at this higher resolution is highly correlated to the underlying chromatin distribution. Heavy concentrations of lamin B directly "cap" the surface of envelope associated, large-scale chromatin domains.     

Correlative single‐molecule localization microscopy and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single‐molecule photo‐switching are opposed. Here, we developed a novel superCLEM workflow that combines triple‐color SMLM (dSTORM & PALM) and electron tomography using semi‐thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub‐compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.     

From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

Background

In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM).

Methodology

To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression.

Conclusion

Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.     

Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles.

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.     

A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.     

Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots

The importance of locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although light microscopy (LM) has been extensively used for mapping protein localization, many studies require the additional resolution of the electron microscope. Here we report the application of small nanocrystals (Quantum dots; QDs) to specifically and efficiently label multiple distinct endogenous proteins. QDs are both fluorescent and electron dense, facilitating their use for correlated microscopic analysis. Furthermore, QDs can be discriminated optically by their emission wavelength and physically by size, making them invaluable for multilabeling analysis. We developed pre-embedding labeling criteria using QDs that allows optimization at the light level, before continuing with electron microscopy (EM). We provide examples of double and triple immunolabeling using light, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.     

High data output and automated 3D correlative light-electron microscopy method

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.     

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.     

Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.     

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80?K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.     

In silico optimization of radioluminescence microscopy

Radioluminescence microscopy (RLM) is a high‐resolution method for imaging radionuclide uptake in live cells within a fluorescence microscopy environment. Although RLM currently provides sufficient spatial resolution and sensitivity for cell imaging, it has not been systematically optimized. This study seeks to optimize the parameters of the system by computational simulation using a combination of numerical models for the system's various components: Monte‐Carlo simulation for radiation transport, 3D optical point‐spread function for the microscope, and stochastic photosensor model for the electron multiplying charge coupled device (EMCCD) camera. The relationship between key parameters and performance metrics relevant to image quality is examined. Results show that Lu₂O₃:Eu yields the best performance among 5 different scintillator materials, and a thickness: 8 μm can best balance spatial resolution and sensitivity. For this configuration, a spatial resolution of ~20 μm and sensitivity of 40% can be achieved for all 3 magnifications investigated, provided that the user adjusts pixel binning and electron multiplying (EM) gain accordingly. Hence the primary consideration for selecting the magnification should be the desired field of view and magnification for concurrent optical microscopy studies. In conclusion, this study estimates the optimal imaging performance achievable with RLM and promotes further development for more robust imaging of cellular processes using radiotracers.      

Electron microscopy of lung rapidly frozen under controlled physiological conditions

Light microscopy of lung rapidly frozen under controlled physiological conditions has been very successful in correlating pulmonary structure and function. However, to study some aspects of pulmonary capillary morphology, the higher resolution of electron microscopy (EM) is necessary. To date, most EM of lung has involed the instillation of a fixative through the airways or vascular system, techniques that probably alter the normal pressure relationships of the capillaries and therefore their morphology. We describe here a technique for rapidly freezing lung to a depth of 1--2 mm below the pleural surface and preparing sections for EM. Lungs from open-chest rats were frozen at various transpulmonary pressures with cold (--80 degrees C) 70% ethylene glycol. Small pieces were then fixed with a solution containing glutaraldehyde and paraformaldehyde for 24 h at --50 degrees C. Staining was with osmium tetroxide and uranyl acetate. Lung frozen at high volumes showed marked stretching of the alveolar septa with severe deformation of the capillaries. Lung frozen at low inflation pressures revealed open capillaries containing numerous red blood cells; in addition, infolding of the alveolar wall was frequently seen. We conclude that this technique gives a level of preservation of rapidly frozen lung suitable for electron microscopy.     

Integrative structure and function of the yeast exocyst complex

Exocyst is an evolutionarily conserved hetero‐octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative‐stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride cross‐links from chemical‐crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo‐EM structure, cross‐links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo‐EM structure, except for Sec5. While not observed in the cryo‐EM map, the integrative model localizes the N‐terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low‐resolution structural data can inform biologically relevant hypotheses, even in the absence of high‐resolution data.     

Fully hydrated yeast cells imaged with electron microscopy

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells.     

From micro to nano: recent advances in high-resolution microscopy

Improving the spatial resolution of optical microscopes is important for a vast number of applications in the life sciences. Optical microscopy allows intact samples and living cells to be studied in their natural environment, tasks that are not possible with other microscopy methods (e.g. electron microscopy). Major advances in the past two decades have significantly improved microscope resolution. By using interference and structured light methods microscope resolution has been improved to approximately 100 nm, and with non-linear methods a ten times improvement has been demonstrated to a current resolution limit of approximately 30 nm. These methods bring together old theoretical concepts such as interference with novel non-linear methods that improve spatial resolution beyond the limits that were previously assumed to be unreachable.