JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 8:217-222 (1988)

A Study of Section Wrinkling on Single-Hole Coated Grids Using TEM and SEM

ANDRE ABAD Laboratory of Electron Microscopy and Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108

KEY WORDS Formvar film

ABSTRACT To prevent section wrinkles usually encountered with the use of coated single-hole grids, a simple method was developed. Formvar film resting on a platform with holes (3.5 mm diameter) was heated with a slide warmer (60-65OC). The bottom of a glass petri dish was inverted over the platform to keep the ambient air at the desired temperature. Sections were picked up from the boat of the diamond knife with a single-hole grid and deposited at the orifice of the platform and allowed to dry. The grids were then carefully pushed through the orifice of the platform with the blunt head of a nail (3 mm diameter).

INTRODUCTION

Wrinkling and folding of thin sections pose a major problem in electron microscopy when using Formvar-coated single-hole copper grids. If the area of interest is located on a small surface of a single section, a mesh grid is adequate. However, studies involving se- rial sectioning or low-magnification or ste- reological studies do not carry this option, and the use of a single-hole grid with sup- porting film is imperative. In the study of Pikakaski and Suoranta (1985), low-viscosity epoxy resin was recommended to eliminate the wrinkles. The use of low-viscosity resins such as Spurrs (Spurr, 1969) and Quetol 651 (Kushida, 19741, however, do not eliminate the problem from many specimens. The sam- ples were often distorted with wrinkles, mak- ing the grid unusable. Since the occurrence of wrinkles does not seem to be a function of the viscosity of the resins, other parameters needed to be investigated (e.g., hardness of the block, chloroform vapor, heat treatment). Wrinkles in sections mounted on Formvar occur erratically both in frequency and de- gree of severity from section to section. The present study involved the elaboration of a protocol that would consistently prevent wrinkles throughout the sample regardless of the type of sample or resin used. A white granular leukocyte (granulocyte) represent- ing a single animal cell with large nuclei and sound and decayed xylem of deciduous and coniferous wood were chosen to study.

MATERIALS AND METHODS The Formvar was obtained by preparation

of a solution of 0.35% Formvarlethylene di- chloride solution. A slide, cleaned with 95% ethanol, was dipped twice in this solution, with drying between dips. The edges of the slide were scraped with a razor blade and put into a freezer for 5-8 min to facilitate the separation of the film from the slide. The film was floated off on glass-distilled water by submerging the slide at a 30" angle. An alu- minum platform (2.5 x 5.5 x 1.5 cm height) with holes (3.5 mm diameter) was used to pick up the film (Fig. la).

The platform was placed on a slide warmer until dry, with a petri dish inverted over it to reduce the settling time. After sectioning, some sections were mounted on uncoated mesh grids; others were put on Formvar films (on the platform) and dried at room tempera- ture. The rest of the sections were placed on Formvar films (on the platform) warmed un- der the petri dish to different temperatures.

The dull sides of copper, single-hole grids were coated with 0.5% Formvar by dipping them into the solution and blotting the shiny side on filter paper (Fig. lb). Using this

This is paper No. 15,429, Scientific Journal Series, Minnesota Agriculture Experiment Station.

Received June 8, 1987; accepted July 28, 1987. Address reprint requests to Andre Abad, Laboratory of Elec-

tron Microscopy and Department of Plant Pathology, 495 Bor- laug Hall, 1991 Buford Circle, University of Minnesota, St. Paul MN 55108.

0 1988 ALAN R. LISS, INC

2 18 A. ABAD

Fig. 1. a: The Formvar film was floated off a slide (cleaned with a 95% ethanol solution) and is picked up with the clean platform. b: The single-hole grids dipped into a 0.5% Formvariethylene dichloride solution are blotted (dull side up) on a paper filter for adhesion on the platform film (the oval orifices of the grids are free of the Formvar). c: The sections are picked up with single-hole grids. d: The grids containing the trapped sections are

deposited at the orifice of the platform (dull side down), which, has been and will remain throughout on the slide warmer except for during the collecting of the grids. e: The loaded platform (one grid or several grids at a time) is returned to the slide warmer and allowed to dry (5-10 mid. f: The grids are pushed through the orifice of the platform onto a filter paper and are collected.

STUDY OF SECTION WRINKLING 2 19

method, grids adhered to the film. After the sections were picked up (Fig. lc), the grids were deposited, dull side down, over the hole of the platform (Fig. Id). Two epoxy resins with low viscosity were used, Spurrs and Quetol 651. Once the grids had dried (Fig. le), they were pushed through the orifice of the platform onto a filter paper with the blunt head of a small-diameter nail (Fig. 10. Gran- ulocytes were fixed in 2.5% glutaraldehyde cacodylate buffer at pH 7.2 for 2 hr, rinsed twice with the buffer (10 min), treated for 1 hr in 1% osmium buffer solution, and rinsed twice for 10 min each with double-distilled water, followed by 1 hr in 0.5% aqueous ur- any1 acetate solution (in block staining). They were dehydrated in an acetone series and embedded in Quetol media (Kushida, 1974). Sections of granulocytes were stained with 5% uranyl acetate (5 min) and Coggeshell's lead citrate (2 rnin). Wood samples were fixed with 1% aqueous potassium permanganate for 10 min, rinsed with distilled water twice, dehydrated in an ethanol series, and embed- ded in Spurrs or dehydrated with a Quetol 651 resin series (residwater) and embedded in Quetol. The sections, 100-150-nm thick, were cut with a Reichert Ultracut E micro- tome and observed with a Hitachi H-600 transmission electron microscope (sections 60-80 nm for routine EM as well as 0.1 pm €or X-ray analysis are also subject to wrin- kling). Sections obtained from Quetol651 and Spurrs were treated with chloroform vapors both on the boat and on the platform. The grids, after TEM observations, were then coated with gold with a sputter-coater and viewed for SEM with a Hitachi S-450. They were placed on the center of the stub without tape or paint.

RESULTS

Sections were subject to chloroform vapors both on the boat of the diamond knife and on the platform. Although the Spurrs sections expanded substantially and the Quetol sec- tions did not as a property of the resin, the se- vere wrinkling was not corrected by chloroform treatment (Harris, 1978; Som- mer et al., 1979; Millonig, 1980). The sections mounted on uncoated 200-mesh grids were wrinkle-free, whereas similar sections mount- ed on Formvar single-hole grids were severely wrinkled. Wrinkles appeared to form during settling of the sections onto the Formvar film. The observation agreed with the inefficacy of the chloroform vaDor treatment of coated sin- gle-hole grids. -

The variation in the infiltration and embedment times of both resins had no effect on the reduction of the wrinkles. When dif- ferent formulations of Quetol were used to increase the hardness of the resin, no dif- ference in the number of wrinkles was ob- served. The wrinkles were always absent in the sections from the mesh grids and the sections on single-hole grids that were dried on the warming plate but were always pres- ent in varying degrees of severity in the sin- gle-hole grids dried at room temperature. The minimum effective temperature best for the granulocyte was 60"; for woody tissue it was 65°C. The film was not affected by the heat and humidity inside the petri dish. The heat- dried Formvar film was also as stable under the electron beam as the films dried at room temperature.

A section of granulocytes dried at room temperature, with severe wrinkles located in the nuclei, is shown in Figure 2a. The large arrow indicates the direction of the cut. The wrinkles do not have any particular orienta- tion associated with the preparation of the section. The wrinkles may be straight or can assume an "S" shape (small arrow). Sections dried with heat (60°C) were wrinkle-free (Fig. 2b). Under close examination, the wrinkles seem to start and end wtih a Y configuration (Fig. 2c, arrow). Under the electron micro- scope beam, they are recognizable by a dark line inhibiting any ultrastructural details and slightly distorting the specimen along its boundary. They can be found in either the cell or the blank resin or in both. A granulo- cyte after heat treatment, free of wrinkles, is shown in Figure 2d.

The wrinkles are larger when located in the blank resin than those found in the cells. The area of specimen affected, such as cell wall or nuclei, restricted their length and possibly their appearance. In sections of birch wood (Fig. 2e), the wrinkles were often asso- ciated with the large secondary wall of the fiber cells. The heat-treated sections of birch are shown in Figure 2f. The micrographs (e,0 are of samples embedded with Spurrs, a low- contrast resin.

Figure 3a is a sample section of pine wood with no tilt of the stage (SEMI. The wrinkles, viewed from above, appear flat, with a dark line (tangent to the tube) in the middle (ar- row), showing the location where the section adheres to itself and the Formvar film. When the SEM stage was tilted to a 45" angle, the wrinkles revealed their tubular shape Fig. 3b).

220 A. ABAD

Fig. 2. a,b: Sections of single animal cells with large nuclei (granulocytes) showing the severe wrinkles in the section dried at room temperature (a) and heat-treated sections without wrinkles (b). See text for details. ~ 6 3 0 . c: Severe wrinkles located on the nucleus in a single granulocyte. The section was dried at room temperature.

See text for details. X8,OOO. d A granulocyte free of wrinkles, representing the results obtained with heat treatment. X8,OOO. e,f: Sample of deciduous wood with wrinkles located in the secondary cell wall when dried at room temperature (el and section treated with heat without wrinkles (0. x520.

Fig. 3. a: Scanning electron micrograph of a section of a section from coniferous wood showing the shape of of coniferous wood revealing the location where the sec- the wrinkle. The specimen was observed at a 45" tion and the Formvar film meet. Observed without tilt- angle. X 12,000. ing the stage. X7,800. b: Scanning electron micrograph

222 A. ABAD

DISCUSSION

A relation between specimen type and wrinkle formation seems to be implied by the fact that some specimens rarely have wrin- kles whereas others always have severe wrinkles (Pikakaski and Suoranta, 1985). Thin sections of biological material do not have uniform density. Denser areas will touch and bond to the film first, preventing the adjacent areas of the section from obtain- ing the surface required to settle flat. The lack of film surface available for the section to settle on causes wrinkles to form. Samples seldom found with wrinkles can be inter- preted as having a lack of excessive variation of density in the specimens. As a result, wrinkles are not produced by every section preparation, nor do they have any predeter- mined orientation, except when the structure of the sample produces it.

The morphology of the wrinkles was inves- tigated with the scanning electron micro- scope. They have a tubular shape, and the outside envelope is uniform. One explanation for the tubular shape involves the role of the water. As two section areas make contact with the film and attached themselves to it, they predetermine the film surface available to the area of the section between them. The difference in the two areas determines the size of the wrinkle. The water under the sec- tion allows the section to settle as it evapo- rates from the points of contact. When the section runs out of the area available and touches itself, the water has determined the tubular shape of the wrinkle. As indicated by the micrographs (Figs. 2e,3a), the forma- tion of several wrinkles can result from only two areas touching the film first. It appears that the orifice of the tube sealed itself out (the water is evaporated) by collapsing the top, creating the Y shape observed at both ends of the wrinkle (arrow in Fig. 212).

The warming of the film and of the section seems ultimately to be directed toward the evaporation of the water. The heat expands the sections, and, with a faster evaporation of the water, the settling effect of the denser areas of the cell within the section is nulli- fied or greatly reduced. The section settles faster and straighter.

This simple technique has been found to alleviate wrinkles consistently in animal and plant cells. Other investigations in our labo- ratory using a wide variety of samples with Epon 812, Epon-araldide, soft and hard for- mulations of Spurrs, Quetol 651, and LR white resins also indicate that wrinkles are not encountered when this technique is used.

ACKNOWLEDGMENT

The author thanks Dr. Robert A. Blan- chette and Rod Kuehn for their support and review of the manuscript.

REFERENCES

Burke, C.N. and Geiselman, C.W. (1971) Exact anhy- dride epoxy percentages for electron microscopy embedding (Epon). J. Ultrastruct. Res. 36:119.

Harris, W.M. (1978) Flattening and staining semithin epoxy sections of plant material. Stain Technol. 53:298-300.

Kushida, H. (1974) A new method for embedding with a low viscosity epoxy resin ‘Quetol 651’ (letter). J . Elec- tron Microsc. 23:197.

Millonig, J . (1980) How to avoid wrinkles while staining thick plastic sections. Stain Technol. 55:118-119.

Pikakaski, K., and Suoranta, U.-M. (1985) Effects of dif- ferent epoxies on avoiding wrinkles in thin sections of biological specimens. J. Electron Microsc. Technol. 2:7-10.

Sommer, J.R., Taylor, I., and Scherer, B. (1979) Wrinkle- free thick sections of tissue embedded in hard plastics. Stain Technol. 54:106-107.

Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J . Ultrastruct. Res. 26:31-43.