Freeze-substitution Method Research Articles

Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx.

Simple SummaryThe glycocalyx (GCX) is a hydrated, gel-like layer of biological macromolecules attached to the cell membrane. The GCX acts as a barrier and regulates the entry of external substances into the cell. The function of the GCX is highly dependent on its structure and composition. Pathogenic factors can affect the protective structure of the GCX. We know very little about the three-dimensional organization of the GXC. The tiny and delicate structures of the GCX are difficult to study by microscopic techniques. In this study, we evaluated a method to preserve and label sensitive GCX components with antibodies for high-resolution microscopy analysis. High-resolution microscopy is a powerful tool because it allows visualization of ultra-small components and biological interactions. Our method can be used as a tool to better understand the role of the GCX during the development and progression of diseases, such as viral infections, tumor invasion, and the development of atherosclerosis.The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.

Stages of the symbiotic zooxanthellae–host cell division and the dynamic role of coral nucleus in the partitioning process: a novel observation elucidated by electron microscopy

The relationship of the symbiotic algae or commonly referred as zooxanthellae (zo) and its host invertebrate species is uniquely observed among cnidarians where these tiny single-celled endosymbionts are used to reside densely within the gastrodermal tissue. However, at the ultrastructural level, little has been known on how zo are maintained through proliferation within its host cells. In the present study, the stages of division of the zo that occurred in the coral cells were investigated by electron microscopy. Scleractinian Pocillopora damicornis was fixed by freeze-substitution methods using a high-pressure freezer and embedded in resin. Ultrathin sections were observed using transmission electron microscope. Symbiotic cell at interphase showed the cortical fine structures of the non-dividing zo enclosed by the outermost plasma membrane (zpm). Overlying them, two distinct layers of intermediate membranes (inm) and a symbiosome membrane (sm) were found. When cytokinesis occurred, the zpm at the opposite division regions formed ‘U-shaped’ furrows which led amphiesmal vesicle (av) to protrude inwardly. As they invaginates, new avs and series of cortical microtubules (mi) are used to develop concomitant with the symbiont’ nuclear division. After the cytokinesis, the overlying membranes successively invaginate; thus, the daughter zooxanthellae were transformed into coccoid cells. These cells, however, remain contiguous to each other until when the coral nucleus (cnu) migrated toward one of the cleavage sites to intervene and dissociate them. Upon occupying the interzooxanthellar space, host nuclear division occurred prior to cytokinesis. These events involving the role of cnu in the partitioning process are first reported in this study, in which its dynamic intervening behavior had been elucidated as a novel finding from the cnidarian species.

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells.

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

Electron Tomography Revels that Milk Lipids Originate from Endoplasmic Reticulum Domains with Novel Structural Features.

Lipid droplets (LD) are dynamically-regulated organelles that originate from the endoplasmic reticulum (ER), and function in the storage, trafficking and metabolism of neutral lipids. In mammary epithelial cells (MEC) of lactating animals, intact LD are secreted intact into milk to form milk lipids by a novel apocrine mechanism. The secretion of intact LD and the relatively large amounts of lipid secreted by lactating MEC increase demands on the cellular processes responsible for lipid synthesis and LD formation. As yet these processes are poorly defined due to limited understanding of LD-ER interactions. To overcome these limitations, we used rapid-freezing and freeze-substitution methods in conjunction with 3D electron tomography and high resolution immunolocalization to define interactions between LD with ER in MEC of pregnant and lactating rats. Using these approaches, we identified distinct ER domains that contribute to lipid droplet formation and stabilization and which possess unique features previously unrecognized or not fully appreciated. Our results show nascent lipid droplets within the ER lumen and the association of both forming and mature droplets with structurally unique regions of ER cisternae, characterized by the presence of perilipin-2, a protein implicated in lipid droplet formation, and enzymes involved in lipid synthesis. These data demonstrate that milk lipids originate from LD-ER domains with novel structural features and suggest a mechanism for initial droplet formation in the ER lumen and subsequent maturation of the droplets in association with ER cisternae.

Endosymbiosis-related changes in ultrastructure and chemical composition of Chlorella variabilis (Archaeplastida, Chlorophyta) cell wall in Paramecium bursaria (Ciliophora, Oligohymenophorea)

Chlorella variabilis, a symbiotic alga, is usually present in the cytoplasm of Paramecium bursaria, although it can be cultured in host-free conditions. Morphological and chemical properties of its cell wall were compared between its free-living and symbiotic states. Transmission electron microscopy (quick-freezing and freeze-substitution methods) revealed that the cell wall thickness of symbiotic C. variabilis was reduced to about half that of the free-living one. Chemical properties of the cell wall were examined by treatment with three fluorescent reagents (calcofluor white M2R, FITC-WGA, and FITC-LFA) having specific binding affinities to different polysaccharides. When the algae were re-introduced into Paramecium host cells, calcofluor fluorescence intensity reduced by about 50%. Calcofluor stains β-d-glucopyranose polysaccharides such as cellulose, N-acetylglucosamine, sialic acid, and glycosaminoglycans. Because treatment with cellulase showed no effect on calcofluor fluorescence intensity, we consider that cellulose is not majorly responsible for the stainability of calcofluor. Staining intensities of FITC-WGA and FITC-LFA were similar in the free-living and symbiotic conditions, suggesting that N-acetylglucosamine and sialic acid are also not responsible for the reduction in the stainability of calcofluor associated with intracellular symbiosis. The amount of glycosaminoglycans on the cell wall may decrease in C. variabilis present in the cytoplasm of P. bursaria.

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy.

Despite all the technological advances at the light microscopylevel, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.

In recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. Vaccinia virus, a member of the Poxviridae family, presents one of the most complex viral structures. The classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. This classical view was challenged by Peters and Muller (1963), who demonstrated the presence of a folded tubular structure inside the virus core and stated the difficulty in visualizing this structure, possibly because it is labile and cannot be preserved by conventional sample preparation. Therefore, this tubular structure, now called the nucleocapsid, has been mostly neglected over the years. Earlier studies were able to preserve the nucleocapsid, but with low efficiency. In this study, we report the protocol (and troubleshooting) that resulted in preservation of the highest numbers of nucleocapsids in several independent preparations. Using this protocol, we were able to demonstrate an interdependence between the formation of the virus core wall and the nucleocapsid, leading to the hypothesis that an interaction exists between the major protein constituents of these compartments, A3 (core wall) and L4 (nucleocapsid). Our results show that high pressure freezing and freeze substitution can be used in more in-depth studies concerning the nucleocapsid structure and function.

Hyphal tip cytoplasmic organization in four zygomycetous fungi

We have examined the hyphal tip structure in four zygomycetous fungi: Mortierella verticillata (Mortierellales), Coemansia reversa (Kickxellales), Mucor indicus and Gilbertella persicaria (Mucorales) using both light and transmission electron microscopy. We have used cryofixation and freeze-substitution methods to preserve fungal hyphae for transmission electron microscopy, which yielded improved preservation of ultrastructural details. Our research has confirmed studies that described the accumulation of secretory vesicles as a crescent at the hyphal apex (i.e. the apical vesicle crescent [AVC]) and provided a more detailed understanding of the vesicle populations. In addition, we have been able to observe the behavior of the AVC during hyphal growth in M. indicus and G. persicaria.

Rapid Embedding Methods into Epoxy and LR White Resins for Morphological and Immunological Analysis of Cryofixed Biological Specimens

A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5-2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.

Ultrastructural observation of natural field phytoplankton cells by using rapid freezing and freeze substitution

Transmission electron microscopy (TEM) is a basic technique used to study the ultrastructures of phytoplankton. However, the use of chemical fixation to prepare phytoplankton samples for TEM is laborious and time-consuming. Furthermore, chemical fixation often causes artificial structural changes (artifacts) in phytoplankton cells. In this study, we induced and optimized a rapid freezing and freeze substitution (RFS) method, accompanied by a filter dehydration system, to prepare TEM samples of marine microalgae, including Heterosigma akashiwo, Chattonella antiqua (Raphidophyceae), Heterocapsa circularisquama (Dinophyceae), Chaetoceros tenuissimus (Bacillariophyceae), and Teleaulax amphioxeia (Cryptophyceae). The whole procedure was much more efficient than the traditional method. In addition, the fine structure of each microalgal cell was well preserved. For example, the fine structures of the nuclear membrane, nuclear pores, and several vesicles were clearly observed, which were comparatively difficult to observe using the chemical fixation method. In addition, this new technique was applicable to the natural blooming cells of H. akashiwo, and high-resolution ultrastructural images were successfully observed. This is the first report on the application of the modified-RFS method for natural phytoplankton assemblages. This method can be used for diverse phytoplankters in natural waters, and the high-quality observations are expected to provide useful information toward understanding phytoplankter physiology and ecology.

Applicable technical method for lower temperature freeze-substitution of cryo-fixed cells

Cryo-fixation and freeze substitution followed by microscopy are commonly used sample preparation methods for visualizing the morphology of intracellular organelles. Freeze substitution is an especially important preparative step because it enables the preservation of intracellular structures in cryo-fixed cells close to the living states for visualization. In this study, we describe a novel freeze substitution method and device that uses both dry ice and liquid nitrogen as coolants rather than dry ice alone. We found that our approach achieved lower temperatures for longer periods of time than conventional devices and therefore allows major improvements in preservation quality, ultimately facilitating high contrast imaging of intracellular organelles. The technical approaches that are realized by this valuable approach are simple, rapid, and applicable for visualizing cellular events of interest.

Ultrastructural Analysis of Lipid Incorporation in the Embryonic Silkworm,Bombyx mori

Insect eggs store many lipid droplets as an energy source for embryonic development. We previously reported that lipid droplets are incorporated into embryos in three steps in the silkworm, Bombyx mori. The midgut plays important roles in lipid incorporation during the second and third steps, whereas the manner of lipid incorporation during the first step is still unknown. In this study, we focused on how lipids were incorporated into the embryo in the first step, compared with the mechanisms used in the second step, by means of transmission electron microscopy using the high-pressure freezing and freeze substitution method. At the beginning of the first step (blastoderm formation stage), some lipid droplets were observed in each cell of the embryonic tissues. Lipid droplets were seen to be derived from the oocyte peripheral cytoplasm by superficial cleavage. At the end of the first step (late appendage formation stage), some lipid droplets were attached to the elongated rough endoplasmic reticulum (rER). It seemed that formation of the lipid droplets occurred in embryonic cells at the end of the first step, because the rER is the site of biogenesis of lipid droplets. The incorporation of lipid droplets in the first step may be subdivided into two stages: the blastoderm formation stage and the subsequent stage before blastokinesis.

The effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bone

The effects of cryopreservation on tendon allograft have been reported, but remain unclear, particularly the potential effects on mechanical properties and histological changes by ice crystal formation. There are also few studies about effects of heating for sterilization of tendon. We evaluated the effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bones (BTBs). BTBs were processed by cryopreservation at -80 degrees C for 3 weeks, or heating at 80 degrees C for 10 min. Tensile testing and histomorphological examination were performed. The cryopreservation of tendons showed less influences on their mechanical properties. When cryopreserved BTBs in frozen state were fixed by freeze-substitution method, many spaces were observed in interfibrillar substances. These results suggest that the collagen fibers of cryopreserved tendons were histomorphologically affected by ice crystals. The heating of tendons completely destroyed the collagen fibers of the tendons and is therefore thought to be inappropriate for the sterilization of BTBs.

Electron microscopic examination of uncultured soil-dwelling bacteria

Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze-substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram-positive type; (iii) Gram-negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram-negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram-negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil-dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low-temperature, low-nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages.

High Pressure Freezing (HPF) using the Compact HPF 01 Unit and Evaluation of Freeze-Substitution Methods for Cyanobacteria and Plant Cells

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

Ultrastructure and Origin of Membrane Vesicles Associated with the Severe Acute Respiratory Syndrome Coronavirus Replication Complex

The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

A Novel Arabidopsis Gene Causes Bax-like Lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.

Effects of different fixation and freeze substitution methods on the ultrastructural preservation of ZYMV-infected Cucurbita pepo (L.) leaves

Different fixation protocols [chemical fixation, plunge and high pressure freezing (HPF)] were used to study the effects of Zucchini yellow mosaic virus (ZYMV) disease on the ultrastructure of adult leaves of Styrian oil pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) with the transmission electron microscope. Additionally, different media were tested for freeze substitution (FS) to evaluate differences in the ultrastructural preservation of cryofixed plant leaf cells. FS was either performed in (i) 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate, (ii) 0.01% safranin in anhydrous acetone, (iii) 0.5% glutaraldehyde in anhydrous acetone or (iv) anhydrous acetone. No ultrastructural differences were found in well-preserved cells of plunge and high pressure frozen samples. Cryofixed cells showed a finer granulated cytosol and smoother membranes, than what was found in chemically fixed samples. HPF led in comparison to plunge frozen plant material to an excellent preservation of vascular bundle cells. The use of FS-media such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde led to low membrane contrast and did not preserve the inner fine structures of mitochondria. Additionally, the use of 0.5% glutaraldehyde caused the cytosol to be fuzzy and partly loosened. ZYMV-induced ultrastructural alterations like cylindrical inclusions and dilated ER-cisternae did not differ between chemically fixed and cryofixed cells and were found within the cytosol of infected leaf cells and within sieve tube elements. The results demonstrate specific structural differences depending on the FS-medium used, which has to be considered for investigations of selected cell structures.

Freeze-substitution methods for Ni localization and quantitative analysis in Berkheya coddii leaves by means of PIXE

Leaves of Ni hyperaccumulator Berkheya coddii were chosen as a model to investigate the influence of eight freeze-substitution protocols on the Ni content and distribution. Freeze-substitution of leaf samples cryofixed by high-pressure freezing was carried out in dry acetone, methanol, diethyl ether and tetrahydrofuran. The same substitution media were also used with dimethylglyoxime added as a precipitation reagent. The samples were infiltrated and embedded in Spurr’s resin. Micro-PIXE analysis of Ni concentration and localization, complemented by proton backscattering for matrix assessment, was performed using the nuclear microprobe at Materials Research Group, iThemba LABS, South Africa. True elemental maps and concentrations were obtained using GeoPIXE-II software. The results were compared with the control results obtained for the parallel air-dried samples, corrected for the water content.The highest Ni content was found in the leaf samples substituted in diethyl ether. This concentration was statistically different from the results obtained for other media. In case of diethyl ether medium Ni was mainly localized in the mesophyll tissue, and the distribution map of this element was in accordance with previous results obtained for freeze-dried and frozen-hydrated leaves of this species. The same distribution pattern was observed for specimens embedded in dry acetone, but Ni concentration was significantly lower. Tetrahydrofuran medium preserved Ni preferentially in the epidermis and vascular tissue, and the elemental map for samples embedded in this medium was distorted. Ni was almost completely washed out from samples substituted in methanol and it was thus impossible to obtain a picture of its distribution. Dimethylglyoxime did not improve the preservation of this element.These results show that diethyl ether is a suitable substitution medium for assessment of Ni concentration and distribution in leaves of B. coddii.

Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae)

Autosporulation is a common mode of propagation for unicellular algae. Autospore-forming species of Chlorellaceae, Chlorella vulgaris Beijerinck, C. sorokiniana Shihira et Krauss, C. lobophora Andreyeva, and Parachlorella kessleri (Fott et Nováková) Krienitz et al. have glucosamine as the main constituent of their rigid cell wall. Recent phylogenetic analyses have showed that the Chlorellaceae divided into two sister groups: the Chlorella-clade and the Parachlorella-clade. We compared the cell wall structure and synthesis of the daughter cell wall in the four species by electron microscopy using rapid freezing and freeze substitution methods. The cell wall of C. vulgaris, C. sorokiniana, and C. lobophora consisted of an electron-dense thin layer with an average thickness of 17-20, 22, and 19 nm, respectively. In these three species, daughter cell wall synthesis occurred on the outer surface of the plasma membrane in the early cell-growth phase. The cell wall of P. kessleri, however, was electron-transparent and 54-59 nm in thickness. Ruthenium red staining of P. kessleri indicated that ruthenium-red-specific polysaccharides accumulated over the outer surface of the plasma membrane. Immunoelectron microscopic observation with an anti-beta-1, 3-glucan antibody and staining with wheat germ agglutinin (WGA) indicated that the cell wall contained beta-1, 3-glucan and WGA specific N-acetyl-beta-D-glucosamine. In P. kessleri, daughter cell wall synthesis began after successive protoplast division. The daughter cell wall synthesis during autosporulation in the four species of Chlorellaceae can be classified into two types-the early and the late types.