Better Epoxy Resin Embedding for Electron Microscopy at Low Relative Humidity

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used: impregnate tissues using the following solutions 1) 70% alcohol—5 minutes, 2) 95% alcohol—4 × 15 minutes, 3) absolute alcohol—3 × 40 minutes, 4) acetone—2 × 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 814,39.4 g; DMP-30, 2.6 g)— 1 hour, 6) acetone-epoxy resin 13—1 hour, 7) epoxy resin—1 hour: complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Poststaining Sections for Electron Microscopy

“Dirt” on electron microscopic sections can generally be avoided by the simple strategem of preventing dry sections from coming in contract with any solution with a dirty surface layer. Wet sections can be pushed through the surface layer of such solutions without ill effect. The first and last solutions to touch a section must be dean, preferably distilled water from a plastic wash bottle.

Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA, nor does it imply its approval to the exclurion of other products that may also be suitable.     

Epon-Maraglas Embedment for Electron Microscopy

There can be little doubt that Epon (Luft 1961) is currently the most widely used embedding medium for electron microscopy. While for the most part this embedding material is reliable, it has the disquieting tendency to fail occasionally to polymerize properly for no apparent reason. to counter this problem, several investigators have proposed that epoxide-anhydride ratios be taken into account to arrive at the best balance of ingredients for the final embedding medium (Coulter 1967, Burke and Geiselman 1971, Chang 1973). in an alternative solution, Mollenhauer (1964) suggested that the Epon be mixed with Araldite, and indeed, this combination has achieved some popularity. Epon-Araldite mixtures, however, have a relatively high viscosity in the unpolymerized state; this may slow permeation of tissue specimens.     

Ultra-Thin Sectioning for Electron Microscopy

A technic is described for obtaining thin sections of animal tissue suitable for electron microscopy. Fixation is accomplished by perfusion of the whole animal with neutral formalin or alcohol formalin followed by immersion of pieces to be examined in neutralized osmium tetroxide. The embedding medium is a mixture of equal parts of n-butyl and ethyl methacrylate polymerized by ultra-violet light. Sectioning is done by means of a glass knife on an International ultra-thin sectioning microtome set at 0.1 μ. The sections are floated on warm water to spread, then placed on Formvar-coated grids, dried, and put into toluene to dissolve the plastic. The technic produces routinely usable, thin sections that show a minimum of damage owing to fixation, embedding, and sectioning.     

Epon-Maraglas Embedment for Electron Microscopy

There can be little doubt that Epon (Luft 1961) is currently the most widely used embedding medium for electron microscopy. While for the most part this embedding material is reliable, it has the disquieting tendency to fail occasionally to polymerize properly for no apparent reason. to counter this problem, several investigators have proposed that epoxide-anhydride ratios be taken into account to arrive at the best balance of ingredients for the final embedding medium (Coulter 1967, Burke and Geiselman 1971, Chang 1973). in an alternative solution, Mollenhauer (1964) suggested that the Epon be mixed with Araldite, and indeed, this combination has achieved some popularity. Epon-Araldite mixtures, however, have a relatively high viscosity in the unpolymerized state; this may slow permeation of tissue specimens.     

Removal of Histological Sections from Glass for Electron Microscopy: Use of Quetol 651 Resin and Heat

A method is described for using the epoxy resin Quetol 651 and heat for convenient and rapid separation of conventional histological sections from glass slides for subsequent ultrathin sectioning for retrospective electron microscopy. The same method is useful when Epon-Araldite is substituted for the Quetol 651 resin.     

Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.     

Hexamethyldisilazane for Scanning Electron Microscopy of Gastrotricha

We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 μm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermeila) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD.     

Cryo-Preservation of Roots for Scanning Electron Microscopy

Fully hydrated roots can be examined in the scanning electronmicroscope after cryo-preservation. Shrinkage associated withdehydration by freeze-drying or critical point drying, to whichroot hairs and secreted mucigel are particularly vulnerable,is avoided. Roots, Lepidium sativum, scanning electron microscopy, cryo-preservation, fully hydrated     

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.     

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.