Electron microscopy of the early Caenorhabditis elegans embryo

The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser‐induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time‐resolved fixation to arrest development at specific stages. The second method uses high‐pressure freezing of whole worms followed by freeze‐substitution (HPF‐FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high‐resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF‐FS and electron tomography. This procedure combines the advantages of time‐resolved fixation and superior ultrastructural preservation by high‐pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.     

Direct attachment of cell suspensions to high-pressure freezing specimen planchettes

We describe a procedure for high‐pressure freezing (HPF) of cultured cells using the HPF aluminium planchettes as a substrate. Cells are either grown directly on planchettes covered with Matrigel or allowed to attach to poly‐l ‐lysine‐coated planchettes. This method allows for rapid transfer of the cells into the HPF and minimizes physical and physiological trauma to the cells. Furthermore, the yield of well‐frozen cells approaches 100% for every cell type we have tried so far. In this report, we show well‐preserved ultrastructure in mitotic and interphase HeLa cells, isolated gastric parietal cells and isolated gastric glands. Immunogold labelling of H⁺/K⁺‐ATPase is shown in parietal cells of isolated gastric glands embedded in LR White resin. The aluminium planchettes appear to have little effect on cell physiology, as demonstrated by the fact that parietal cells cultured for 24–28 h on the planchettes retain their responsiveness to stimulation with histamine.     

Feasibility of high pressure freezing with freeze substitution after long‐term storage in chemical fixatives

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non‐cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols. Microsc. Res. Tech. 76:942–946, 2013. © 2013 Wiley Periodicals, Inc.     

Fine structure of the ommatidia of the short‐faced scorpionfly Panorpodes kuandianensis (Mecoptera: Panorpodidae)

The fine structure of the ommatidia in males of the short‐faced scorpionfly Panorpodes kuandianensis was investigated using scanning and transmission electron microscopy. The result shows that the compound eyes of male P. kuandianensis are of the apposition type, consisting of over 1700 ommatidia. Each ommatidium is composed of a laminated cornea, a eucone crystalline cone, eight retinula cells, a pair of primary pigment cells, and a group of 16 secondary pigment cells. Along the optical axis of the ommatidium, seven elongated retinula cells contribute their rhabdomeres to a centrally fused rhabdom, which is in tight contact with the proximal end of the crystalline cone, but smaller than the cone end in diameter. The eighth retinula cell is located above the basal lamina and only contributes its rhabdomere to the proximal part of the rhabdom. The microvilli of the rhabdom show an orthogonally‐arranged orientation. The ommatidia of Panorpodidae are more similar to those of Panorpidae than Bittacidae in structure, adding weight to the view that the sister group of Panorpodidae is Panorpidae rather than Bittacidae. Microsc. Res. Tech. 76:862–869, 2013. © 2013 Wiley Periodicals, Inc.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

Blue molybdenum oxides: A stain for light and electron microscopy

Blue molybdenum oxides (molybdenum blues) have been prepared from aqueous phosphomolybdic acid solutions and applied to thin and semi-thin sections of glutaraldehyde-fixed, epoxy-embedded tissues. A light blue colour and high electron opacity were found in mast cell granules, the secretion content of goblet cells, and cytoplasmic granules in Drosophila salivary glands. The possibility that binding of blue molybdenum oxides to polyhydroxylic components accounts for the staining and contrasting reactions in some cell structures is briefly discussed.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Centrifuge polarizing microscope. I. Rationale, design and instrument performance

We first describe early uses of the centrifuge for deciphering physical properties and molecular organization within living cells, as well as the development and use of centrifuge microscopes for such studies. The rationale for developing a centrifuge microscope that allows high‐extinction polarized light microscopy to observe dynamic fine structures in living cells is next discussed. We then describe a centrifuge polarizing microscope (CPM) that we developed for observing fine structural changes in living cells which are being exposed to up to ≈ 11 500 times earth's gravitational field (g). With the specimen housed in a rotor supported on an air spindle motor, and imaged through an external microscope illuminated by a precisely synchronized flash of less than 10 ns duration from a Nd:YAG laser, the image of the spinning object remains steady up to the maximum speed of 11 700 rev min^?1, or up to ≈ 11 500 × g. The image is captured, at up to 25 frames s^?1, by an interference‐fringe‐free CCD camera that is synchronized to the centrifuge rotor. At all speeds (in 100 rev min^?1 increments), the image is resolved to better than 1 µm, while birefringence of the specimen, housed in a specially designed specimen chamber that suffers low‐stress birefringence and prevents leakage of the physiological solutions, is detected with a retardance sensitivity of better than 1 nm. Differential interference contrast and fluorescence images (532 nm excitation) of the spinning specimen can also be generated with the CPM. The second part of this study (Inouéet al., J. Microsc. 201 (2001) 357–367, describes several biological applications of the CPM that we have explored. Individual live cells, such as oocytes and blood cells, are supported on a sucrose or Percoll density gradient while other cells, such as cultured fibroblasts and Dictyostelium amoebae, are observed crawling on glass surfaces. Observations of these cells exposed to the high G fields (centripetal acceleration/g) in the CPM are yielding many new results that lead to intriguing questions regarding the organization and function of fine structures in living cells and related quasi‐fluid systems.     

Optimal preservation of the shark retina for ultrastructural analysis: An assessment of chemical, microwave, and high-pressure freezing fixation techniques

Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum, obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent. Microsc. Res. Tech. 75:1218–1228, 2012. © 2012 Wiley Periodicals, Inc.     

High-pressure freezing in the study of animal pathogens

High‐pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot‐and‐mouth disease virus and African swine fever virus by the use of high‐pressure freezing of infected cells. Foot‐and‐mouth disease virus particles are not detected in sections of conventionally immersion‐fixed infected cells, but when the cells are prepared by high‐pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high‐pressure freezing of virally infected cells: first, two sapphire discs frozen ‘face to face’ with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro, and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high‐pressure freezing followed by freeze‐substitution in acetone with uranyl acetate allows high‐sensitivity immunolabelling for these proteins.     

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog ( Rana pipiens) retina was sliced at a thickness of 150 or 350 μm, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO₄ and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-μm slices or 15% low-MW dextran for 350-μm slices) were required for adequate freezing.
The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-μm slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers.
This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.     

High-pressure freezing of plant cells cultured in cellulose microcapillaries

A new microculturing technique for plant cells was used to meet the requirements of high-pressure freezing (HPF). The plant cells were cultured inside cellulose microcapillaries, providing an easy-to-handle method for a real in situ fixation. The high viability of the cells was demonstrated by regenerating shoots from microcalluses cultivated by this method. In general, the freezing quality of the high-pressure frozen samples was excellent across the whole diameter of the capillaries, as shown with ultrathin sectioned cells after freeze-substitution and embedding in Spurr's resin. In comparison with conventional chemically fixed cells, cultured under identical conditions, all membranous compartments and organelles were more turgid and smoother after HPF. The cytoplasm and the matrix of the organelles were more homogeneous and dense. Thus, high-pressure freezing in combination with the microculture method described here appears to preserve the ultrastructure of chemically untreated plant cells close to the native state.     

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H⁺/K⁺‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H⁺/K⁺‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.     

Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.     

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers

A Nd‐YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament‐dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR‐laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 µm but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament‐mediated processes.     

A simple ethanol-based freeze-substitution technique for marine invertebrate embryos which allows retention of antigenicity

A simple technique has been developed for flash freezing and freeze substituting small (0.5 mm) marine embryos, which effectively preserves both cellular and extracellular components, using inexpensive equipment that is readily available in most laboratories. To achieve this, embryos of the starfish, Pisaster ochraceus, were isolated on copper freeze-fracture EM grids. The embryos were then rapidly frozen by plunging the grids into a supercooled liquid cryogen, and stored in liquid nitrogen. Freeze substitution was carried out by placing the specimens in sealed vials containing anhydrous ethanol at ?90°C for 4–5 days. Following substitution, the specimens were passively warmed to ?20°C over 2 h and then to room temperature over a further 2 h. They were then embedded in either JB4 for light microscopy or Epon or LR White resins for transmission electron microscopy. Four different liquid cryogens, Freon 12, ethane, propane, and nitrogen slush, were tested. Freezing in propane, the best cryogen of the four, gave good preservation of the embryonic cells but poor preservation of the extracellular matrix (ECM). To overcome this, the embryos were exposed to four cryoprotective agents, dimethylsulphoxide, glycerol, ethylene glycol and propylene glycol prior to freezing, and the results were assessed. The experiments demonstrated that good preservation of both cells and ECM could be achieved by adding 15% propylene glycol in sea water to the embryos prior to freezing in propane. Material preserved in this manner not only gave excellent morphological results, but the antigenicity of both native antigens of ECM components and antibodies to which the animals had been exposed in vivo were retained. The application of this technique to other tissues and embryos should prove useful in many future studies.     

Multichannel wide‐field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter‐based multichannel wide‐field microscope for ExEm unmixing‐based FRET imaging (m‐ExEm‐spFRET) containing a constant system correction factor (f_sc) for a stable system. We performed m‐ExEm‐spFRET with four‐ and two‐wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (R_C). We also performed m‐ExEm‐spFRET imaging for single living cells coexpressing CFP‐Bax and YFP‐Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin‐treated cells when R_C were larger than 1, indicating that staurosporin induced significant oligomerisation.     

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A₂ toxins (PLA₂) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA₂ toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.