Epoxy resin as fixative during freeze-substitution

An alternative protocol for freeze-substitution is described. Araldite/Epon embedding medium (20% in acetone) is first used as a stabilizer (as e.g., OsO(4)) and then as embedding medium. The major components of the Araldite/Epon resin formulation react with proteins and lipids and provide for an excellent preservation and reasonable visualisation of the ultrastructure. The ultrastructural appearance can be deliberately influenced with the standard freeze-substitution procedure [Van Harreveld, A., Crowell, J., 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381-386.] using OsO(4) as stabilizing agent by protocols which degrade cytoplasmic and membrane proteins. Epoxy stabilized and embedded samples may become an important tool to get information about the effects of different reagents and protocols used in freeze-substitution. We believe that an in-depth understanding of the procedures is required to correctly interpret images and to complement studies of dynamic processes by light microscopy with reliable, highly detailed ultrastructural information. The block face of epoxy stabilized samples after ultrathin sectioning is highly suited for the analysis of the ultrastructure by AFM.     

A novel in situ atomic force microscopy imaging technique to probe surface morphological features of starch granules

The application of atomic force microscopy (AFM) for observing iodine complexes in starch has been limited due to limitations including granular sample fixation techniques and possible unintended reactions with embedding materials such as epoxy resins or adhesives. In this paper, a new method is described that employs an optical microscopic technique to ensure that the tip of the AFM is scanning a specified granule without any probe-induced particle movement by the AFM probe motion. The direct sprinkling of samples on a two-sided adhesive tape allows investigations in an in situ environment of the un-embedded starch granule surface and thus provides high-resolution images of granule morphology and phase changes of starches in the presence of humidity and with iodine vapor. These observations demonstrate that this novel in situ AFM imaging technique allows us to visualize the hair-like structures on the surface of granular starches when starches are exposed to iodine vapor under humid environments. This study reveals that the hair-like extensions on the starch granule surfaces are strongly dependent on the organization of the glucan polymers within corn or potato starch.     

Epon Resin Infiltration and Immunogold Labelling of Pituitary Secretory Granules after Cryofixation versus Chemical Fixation

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation + freeze-drying (CF-FD), glutaraldehyde fixation (GF) + chemical dehydration, and no fixation— no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.     

Method for the Prfservation of Polystyrene Latex Beads in Tissue Sections

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation + freeze-drying (CF-FD), glutaraldehyde fixation (GF) + chemical dehydration, and no fixation— no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

A method of relating craniofacial sections to topography in embryos.

The transparent properties of the embedding agent glycol methacrylate facilitate orientation of the complex embryonic craniofacial region. This technique allows for consistent and reproducible section-to-topography orientation. We find it to be a valuable adjunct for envisioning three-dimensional relationships. The contrast of external features of the embryo is enhanced when stained lightly with hematoxylin prior to embedding. The craniofacial region of the embedded embryo is removed with a fine surgical saw and reimbedded. Section-to-topography relationships are readily monitored and documented photographically. Furthermore, it is possible to "preview" sections for symmetry and other considerations of orientation by viewing the cutting face of the block under oblique illumination. A relief image of structures is then visible.     

Imaging biological samples by integrated differential phase contrast (iDPC) STEM technique

Scanning transmission electron microscopy (STEM) is a powerful imaging technique and has been widely used in current material science research. The attempts of applying STEM (annual dark field (ADF)-STEM or annular bright field (ABF)-STEM) into biological research have been going on for decades while applications have still been limited because of the existing bottlenecks in dose efficiency and non-linearity in contrast. Recently, integrated differential phase contrast (iDPC) STEM technique emerged and achieved a linear phase contrast imaging condition, while resolving signals of light elements next to heavy ones even at low electron dose. This enables successful investigation of beam sensitive materials. Here, we investigate iDPC-STEM advantages in biology, in particular, chemically fixed and resin embedded biological tissues. By comparing results to the conventional TEM, we have found that iDPC-STEM not only shows better contrast but also resolves more structural details at molecular level, including conditions of extremely low dose and minimal heavy-atom staining. We also compare iDPC-STEM with ABF-STEM and found that contrast of iDPC-STEM is even further improved, moderately in lower frequency domains while highly with preserving high frequency biological structural details. For thick sample sections, iDPC-STEM is particularly advantageous. It avoids contrast inversion canceling effects, and by adjusting the depth of focus, fully preserves the contrast of structural details along with the sample. In addition, using depth-sectioning, iDPC-STEM enables resolving in-depth structural variation. Our results suggest that iDPC-STEM have the place and advantages within the future biological research.     

The influence of embedding media and fixation on the postembedment ultrastructural demonstration of complex carbohydrates. III. High iron diamine staining for sulfated glycoconjugates

The high iron diamine (HID) method for detection of sulfated complex carbohydrate has been applied directly on thin sections of variably fixed tissues embedded in epoxy and nonepoxy resins. Results with postembedment HID staining in mouse intestinal epithelium are compared to those previously obtained using preembedment methods. Sections from epoxy-embedded tissues have been found to exhibit the weakest staining intensity. Intense, specific staining was obtained in tissues not postfixed with osmium tetroxide and embedded in polystyrene, polyester resins, styrene-methacrylate, and especially the styrene-Vestopal W embedding mixture. Postosmication of tissues abolished HID staining in epoxy resins and the styrene-Spurr's resin embedding mixture, but only reduced the staining intensity in tissues embedded in nonepoxy resins.     

Fine structure of cell wall surfaces in the giant-cellular xanthophycean alga <Emphasis Type="Italic">Vaucheria terrestris</Emphasis>

The mechanical strength of cell walls in the tip-growing cells of Vaucheria terrestris is weakened by treatment with proteolytic enzymes. To clarify the morphological characteristics of the components maintaining cell wall strength, the fine structures of the cell walls, with and without protease treatment, were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations indicated that cellulose microfibrils were arranged in random directions and overlapped each other. Most of the microfibrils observed in the inner surface of the cell wall were embedded in amorphous materials, whereas in the outer surface of the cell wall, microfibrils were partially covered by amorphous materials. The matrix components embedding and covering microfibrils were almost completely removed by protease treatment, revealing layers of naked microfibrils deposited deeply in the cell wall. Topographic data taken from AFM observations provided some additional information that could not be obtained by TEM, including more detailed images of the granular surface textures of the matrix components and the detection of microfibrils in the interior of the cell wall. In addition, quantitative AFM data of local surface heights enabled us to draw three-dimensional renderings and to quantitatively estimate the extent of the exposure of microfibrils by the enzymatic treatment.     

A Method of Relating Craniofacial Sections to Topography in Embryos

The transparent properties of the embedding agent glycol methacrylate facilitate orientation of the complex embryonic craniofacial region. This technique allows for consistent and reproducible section-to-topography orientation. We find it to be a valuable adjunct for envisioning three-dimensional relationships. the contrast of external features of the embryo is enhanced when stained lightly with hematoxylin prior to embedding. the craniofacial region of the embedded embryo is removed with a fine surgical saw and re-imbedded. Section-to-topography relationships are readily monitored and documented photographically. Furthermore, it is possible to “preview” sections for symmetry and other considerations of orientation by viewing the cutting face of the block under oblique illumination. A relief image of structures is then visible.     

不同厚度细胞薄切片对 AFM 图像反差的影响

利用原子力显微镜（ AFM ）观察超薄切片的表面,探索表面形貌与切片厚度、朝向等因素的关系以及对图像反差的影响 . 选择三种不同类型的细胞,培养后按电镜超薄切片法固定、包埋并切片后,将不同厚度的切片区分上下表面转移到云母上, AFM 在空气中以接触模式进行观察 . 结果发现,切片表面细胞相对包埋介质的凸起与凹陷与切片本身的厚度密切相关,并随切片厚度的不同呈现有规律的变化 . 实验统计结果显示这种现象可能具有普遍性 .     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy

We used atomic force microscopy (AFM), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM, but not by AFM. The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.     

The Surface Staining of Embedded Tissues

The staining procedure is based on the theory that the freshly cut surface of embedded material will absorb stain only in the exposed tissue elements, provided that the embedding compound itself will not absorb the staining fluid. Concentrated stains are used for short intervals to insure minimum penetration. For paraffin embedded materials: (1) Cut block, preferably on microtome, to the desired tissue surface. (2) Rinse in absolute alcohol. (3) Float face down in stain. (Ripe, concentrated alum hematoxylin—Galigher's formula recommended—will stain in 10 to IS minutes. Heidenhain's iron hematoxylin works exceptionally well in some cases.) Mordant 20% alum 5 to 10 minutes, briefly rinse, and stain comparable 5 to 10 minutes in 1 to 1.5% hematoxylin. (4) Allow to become blue in tap water (for hematoxylin stains). (5) Counter-stain if desired. (6) Dehydrate in absolute alcohol for not more than 10 minutes. (7) Dry for 15 to 20 minutes. (8) Trim block to 2-3 mm. and mount between two cover glasses by use of microflame. Attach mount to slide with balsam. For celloidin embedded materials: (1) Dehydrate block with 90% alcohol, phenol-toluene, finally pure toluene. (2) Rinse cut surface with 90% alcohol, then apply stain. (3) Wash, after hematoxylin stains, counterstain if desired. (4) Dehydrate surface, 90% alcohol, phenol toluene, pure toluene, and mount in medium dissolved in toluene.

Possible applications of surface staining technic are suggested and illustrated.     

原子力显微镜在双微体形态学研究中的应用

原子力显微术(atomic force microscopy,AFM)是一种新型的纳米显微技术,由于其拥有标本制备简单、分辨率高等优点,因此常用于细胞超微结构的观察。双微体(double minute chromosomes,DMs)是基因扩增的主要表现形式,经常出现在肿瘤细胞及耐药细胞中,可使肿瘤细胞获得生存优势或产生耐药性,因此对双微体进行研究可使人类了解肿瘤的生长特性及其抗药性的产生机理。为寻找一种研究双微体的有效方法,本实验利用原子力显微镜对小鼠耐氨甲喋呤细胞3T3R500中的双微体进行观察,在获得双微体高分辨AFM形态图的同时,还对双微体的大小进行了测量,发现细胞中双微体大小存在差异。此外,就原子力显微镜在双微体研究中的一些技术细节进行了探讨。实验结果表明原子力显微术是研究双微体的一种有效手段。     

Staining of Different Tissues in Thick Epoxy Resin-Impregnated Sections of Human Fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczko and Levai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

Acquiring information of the neural structures in the whole‐brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high‐throughput light‐sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain‐wide distribution of specific neurons or neural circuits.      

Epoxy-Slide Embedment of Cytochemically Stained Tissues and Cultured Cells for Light and Electron Microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cost-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.