Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy

Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.     

Electron tomographic analysis of frozen-hydrated tissue sections

Electron tomography of frozen-hydrated tissue sections enables analysis of the 3-D structure of cell organelles in situ and in a near-native state. In this study, 160-200-nm-thick sections were cut from high-pressure frozen rat liver, and improved methods were used for handling and mounting the sections. Automated data collection facilitated tilt-series recording at low electron dose (approximately 4000 e(-)/nm(2) at 400 keV). Higher doses (up to 10,000 e(-)/nm(2)) were found to increase contrast and smooth out surface defects, but caused section distortion and movement, with likely loss of high-resolution information. Tomographic reconstruction showed that knife marks were 10-40 nm deep and located on the "knife face" of the section, while crevices were 20-50 nm deep and found on the "block face." The interior of the section was normally free of defects, except for compression, and contained useful structural information. For example, the topology of mitochondrial membranes in tissue was found to be very similar to that in frozen-hydrated whole mounts of isolated mitochondria. In rare cases, a 15-nm banding pattern perpendicular to the cutting direction was observed in the interior of the section, most evident in the uniformly dense, protein-rich material of the mitochondrial matrix.     

Electron tomography: towards visualizing the molecular organization of the cytoplasm

Electron tomography of thin sections of plastic-embedded biological material provides valuable insights into the organellar organization of cells. With the advent of automated data-acquisition procedures, it has become possible to study radiation-sensitive specimens, such as frozen-hydrated organelles and whole cells. This opens up exciting perspectives for visualizing the molecular organization of the cytoplasm.     

Application of scanning electron microscopy to x-ray analysis of frozen- hydrated sections. I. Specimen handling techniques

X-ray microanalysis of frozen-hydrated tissue sections permits direct quantitative analysis of diffusible elements in defined cellular compartments. Because the sections are hydrated, elemental concentrations can be defined as wet-weight mass fractions. Use of these techniques should also permit determination of water fraction in cellular compartments. Reliable preparative techniques provide flat, smooth, 0.5 micrometers-thick sections with little elemental and morphological disruption. The specimen support and transfer system described permits hydrated sections to be transferred to the scanning electron microscope cold stage for examination and analysis without contamination or water loss and without introduction of extraneous x- ray radiation.     

Electron tomography of biological samples

Electron tomography allows computing three-dimensional (3D) reconstructions of objects from their projections recorded at several angles. Combined with transmission electron microscopy, electron tomography has contributed greatly to the understanding of subcellular structures and organelles. Performed on frozen-hydrated samples, electron tomography has yielded useful information about complex biological structures. Combined with energy filtered transmission electron microscopy (EFTEM) it can be used to analyze the spatial distribution of chemical elements in biological or material sciences samples. In the present review, we present an overview of the requirements, applications, and perspectives of electron tomography in structural biology.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1497–1505.Original Russian Text Copyright © 2004 by Marco, Boudier, Messaoudi, Rigaud.     

Three-dimensional imaging of biological complexity

Over the past 5 years, thanks to advances in both instrumentation and computational speed, three-dimensional imaging techniques using the electron microscope have been greatly improved in two areas: electron tomography of cell organelles or cell sections and reconstruction of macromolecules from single particles. Ice embedment has brought a breakthrough in the degree of preservation of specimens under close-to-native conditions. The current challenge is to push the resolution of electron tomographic imaging to a point where macromolecular signatures can be recognized within the cellular context. We show first progress toward this goal by examples in two areas of application: the structure of the muscle triad junction and the architecture and fine structure of mitochondria. As techniques of cryo-microtomy are perfected, we hope to be able to apply tomography to high-pressure frozen sections of tissue.     

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.     

The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells

The structure of cellular organelles, in particular those involved in endocytosis, was studied by electron microscopy with hydrated cryosections. In this technique no chemical treatment is used, and the native structure of organelles can be observed in sections viewed at temperatures below -140 degrees C, using a cold stage accessory on the electron microscope. The compartments of the endocytic pathway were prelabeled with gold markers in the living cell, facilitating the identification of different structures in the cryosections. The structure of most identifiable cellular organelles, including those involved in endocytosis, appeared very similar in the hydrated cryosections to that seen after conventional plastic and cryosections of chemically fixed cells. In particular, the internal membranes of the structure we refer to as the prelysosomal compartment (Griffiths et al., Cell 52, 329-341 (1988] could be clearly visualized in these sections indicating that the organization of these membranes is not a consequence of the chemical fixation process.     

Scale-dependent mechanical properties of native and decellularized liver tissue

Decellularization, a technique used in liver regenerative medicine, is the removal of all the cellular components from a tissue or organ, leaving behind an intact structure of extracellular matrix. The biomechanical properties of this novel scaffold material are currently unknown and are important due to the mechanosensitivity of liver cells. Characterizing this material is important for bioengineering liver tissue from this decellularized scaffold as well as creating new 3-dimensional mimetic structures of liver extracellular matrix. This study set out to characterize the biomechanical properties of perfused liver tissue in its native and decellularized states on both a macro- and nano-scale. Poroviscoelastic finite element models were then used to extract the fluid and solid mechanical properties from the experimental data. Tissue-level spherical indentation-relaxation tests were performed on 5 native livers and 8 decellularized livers at two indentation rates and at multiple perfusion rates. Cellular-level spherical nanoindentation was performed on 2 native livers and 1 decellularized liver. Tissue-level results found native liver tissue to possess a long-term Young’s modulus of 10.5 kPa and decellularized tissue a modulus of 1.18 kPa. Cellular-level testing found native tissue to have a long-term Young’s modulus of 4.40 kPa and decellularized tissue to have a modulus of 0.91 kPa. These results are important for regenerative medicine and tissue engineering where cellular response is dependent on the mechanical properties of the engineered scaffold.     

Electron Tomography in Plant Cell Biology

This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensional resolution and improved preservation of the subcellular components. In particular, the combination of cryofixation/freeze substitution and electron tomography have allowed plant cell biologists to image organelles and macromolecular complexes in their native cellular context with unprecedented three-dimensional resolution （4-7 nm）. Until now, electron tomography has been applied in plant cell biology for the study of cytokinesis, Golgi structure and trafficking, formation of plant endosome/prevacuolar compartments, and organization of photosynthetic membranes. We discuss in this review the new insights that these tomographic studies have brought to the plant biology field.     

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post-embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.     

Electron tomography of vitreous sections from cultured mammalian cells

Cryo-electron tomography of appropriately thin, frozen-hydrated biological specimens has excellent potential for investigating the 3D macromolecular architecture of eukaryotic cells and tissues. Since cardiomyocytes are too thick to be visualised in an intact state, we grew immortalised cell line HL-1 to sub-confluency and harvested the cells by enzymatic detachment prior to hyperbaric freezing, ultramicrotomy, and tomography. We improved the efficiency of tomographic acquisition from vitreous cryosections by implementing two new features: (1) fluorescence microscopy at cryogenic temperatures to search for features of interest without expending any of the tolerable electron dose on secondary (non-imaging) tasks, and (2) the use of colloidal gold as fiducial markers. Vital fluorescent staining and subsequent cryo-fluorescence microscopy of vitreous sections were used to localise mitochondria lying in positions suitable for acquiring tilt series, taking into account section flatness, presence of contamination and proximity to grid bars. To provide a simple and robust means of aligning tomograms, we developed a universally applicable protocol for depositing colloidal gold onto vitreous sections, analogous to the method for applying quantum dots described by Masich et al. [Masich, S., Östberg, T., Norlén, L., Shupliakov, O., Daneholt, B., 2006. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. J. Struct. Biol. 156, 461–468]. Tomograms of thin sections (nominal thickness 65–85 nm) of cardiac mitochondria revealed the interconnectivity of cristae and junctions with the inner mitochondrial membrane. In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6–8 nm.     

Template matching as a tool for annotation of tomograms of stained biological structures

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture.     

Radiation dose reduction and image enhancement in biological imaging through equally-sloped tomography

Electron tomography is currently the highest resolution imaging modality available to study the 3D structures of pleomorphic macromolecular assemblies, viruses, organelles and cells. Unfortunately, the resolution is currently limited to 3-5nm by several factors including the dose tolerance of biological specimens and the inaccessibility of certain tilt angles. Here we report the first experimental demonstration of equally-sloped tomography (EST) to alleviate these problems. As a proof of principle, we applied EST to reconstructing frozen-hydrated keyhole limpet hemocyanin molecules from a tilt-series taken with constant slope increments. In comparison with weighted back-projection (WBP), the algebraic reconstruction technique (ART) and the simultaneous algebraic reconstruction technique (SART), EST reconstructions exhibited higher contrast, less peripheral noise, more easily detectable molecular boundaries and reduced missing wedge effects. More importantly, EST reconstructions including only two-thirds the original images appeared to have the same resolution as full WBP reconstructions, suggesting that EST can either reduce the dose required to reach a given resolution or allow higher resolutions to be achieved with a given dose. EST was also applied to reconstructing a frozen-hydrated bacterial cell from a tilt-series taken with constant angular increments. The results confirmed similar benefits when standard tilts are utilized.     

Characterizing Organelles in Live Stem Cells Using Label-Free Optical Diffraction Tomography

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells—namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell—based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.     

Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles

Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.     

Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography

Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optical resolution to image single molecules. In this paper, we describe the use of soft X-ray tomography, a new tool for quantitative imaging of organelle structure and distribution in whole, fully hydrated eukaryotic Schizosaccharomyces pombe cells. In addition to imaging intact cells, soft X-ray tomography has the advantage of not requiring the use of any staining or fixation protocols—cells are simply transferred from their growth environment to a sample holder and immediately cryofixed. In this way the cells can be imaged in a near native state. Soft X-ray tomography is also capable of imaging relatively large numbers of cells in a short period of time, and is therefore a technique that has the potential to produce information on organelle morphology from statistically significant numbers of cells.     

Luminal particles within cellular microtubules

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.