Determination of the thickness of electron microscopy sections

J. ULTRASTRUCTURERESEARCH4, 413-419 (1960)

413

Determination of the Thickness of Electron Microscopy Sections T. ZELANDER and R. EKHOLM

Department of Anatomy, University of Gothenburg, Gothenburg, Sweden Received August 10, 1960 An interference microscopical method of thickness determination for ultramicrotome sections is described. Thicknesses are determined for methacrylate sections before and after exposure to vacuum and electron beam. Exposure to electron beam is observed to occasion a marked loss of section material (approximately 60%). Thicknesses have been measured and compared for sections cut at two different microtome advance rates, 100 ~ and 250 ~. The mean value of the section thickness in the 100/~ series is 326 ~ and in the 250 series 493/~. The thinnest section in the 100 It series is 190 ~. The distortion of the sections, as revealed by comparison between microtome advance and the actual thickness of the sections, is briefly discussed.

The thickness of an electron microscopical section may be defined as the thickness of the layer of material removed from the block at a stroke of the microtome, or as the thickness of a freshly cut and more or less compressed slice of the block. We may also consider the thickness of a section after exposure to bombardment in the electron microscope. In practice it is often most useful to know the first quantity, since it determines within what range of dimensions superposition effects can be expected, and what thickness should be considered in connection with three-dimensional reconstructions of serial sections. The thickness of sections is commonly estimated from their interference colors in reflected light on the liquid on which they are floated in the microtome. Such estimates are reliable to within 100-200 A for sections thicker than 600 A (5, 6). However, the characteristic grayish tone considered useful for electron microscope work is produced by a wide range of thicknesses below 600 A. The first estimates of section thicknesses were made by SjSstrand (11) and by Porter and Blum (7), who calculated the edge thickness of sections from the length of the shadow after shadow-casting at a known angle. Many different methods have since been tried to determine section thickness (for review, see Reimer, 9). One is ellipsometry, i.e., determination of the elliptic polarization of the beam of light reflected by a section illuminated by plane-polarized light (5, 6). Another technique

414

T. Z E L A N D E R A N D R. E K H O L M

makes use of Tolansky's multiple interferometric procedure after evaporation of silver onto the section (1). Williams and Kallman (16) measured the parallax between a point on the upper surface of the section and another point on the lower surface at a stereo tilting angle of ± 30 ° in an electron microscope, and used this value to calculate section thickness. Reimer (8) based his determinations of section thickness on quantitative estimates of electron transmission. Finally, S6chaud et al. (10) calculated the thickness of sections by counting the relative number of phage particles which were intersected once or twice in serial sections, relating this proportion to the known phage dimensions. Williams' and Kallman's (16) as well as Reimer's (8) techniques do not give any information as to the thickness of the layer removed from the block, since exposure to the electron beam changes the thickness of methacrylate sections. Also, exposure to vacuum and evaporation of metals onto the section surfaces may introduce certain errors. Ellipsometry (5, 6) does not suffer from these shortcomings, but is accurate only to + 50 A, and, furthermore, Peachey did not attempt to measure sections less than about 500 A in thickness. On the whole, techniques used heretofore have not been useful for accurate determination of section thickness in the less-than-600 A range, which is the useful range for high-resolution electron microscopy.

PROCEDURE In the present investigation section thickness was determined with the aid of L. P. Johansson's shearing interferometer (4). This apparatus, using an ordinary light microscope, a special illuminator, and an interferometric unit, operates in the following manner. The incident light from the illuminator, designed to produce a beam of parallel rays, passes a polarizer so that a plane wavefront is established through the object. A Savart plate splits this plane wavefront into two perpendicularly polarized wavefronts a small distance apart. These two wavefronts are caused to interfere with one another in a recombining unit, and in the image plane two images appear in interference colors corresponding to the optical path difference between the object and the background. A compensator, consisting of an isotropic glass wedge with a central aperture, is situated in front of the Savart plate. By rotation of the compensator the interference color of the whole field of vision outside a reference area corresponding to this aperture can be controlled. The interference colors of the background and of the object are in turn matched to the color of the reference are and the respective rotational angles of the compensator noted. The difference between the sines of these angles is proportional to the optical path difference between the object and the background. The thickness of the section is calculated according to the formula t = A/(n v -n~), where t = thickness, A = optical path difference (o.p.d.), n~ = refractive index of the object, and n, = refractive index of air. Measurements with this equipment were made on sections of methacrylate embedding medium (85% butyl and 15% methyl methacrylate) cut on two thermal expansion ultramicrotomes without bearings, one microtome of our own construction (13) and one Ultro-

THICKNESS OF E L E C T R O N MICROSCOPY SECTIONS

415

tome. 1 The advance per minute of the microtomes was measured with a dial indicator 2 calibrated in 1000-A units. The sections were cut with glass knives, floated on a 25 % dioxane solution, and left to stretch for about 15 minutes. N o xylene or other agent was used to improve stretching. The sections were picked up on formvar-coated Athene copper grids. The first series of thickness determinations was made on 12 sections cut at a microtome feeding of approximately 200 A per section on the microtome of our own construction. In our experience this feeding rate has been found suitable for routine sectioning of most biological specimens; such sections will give different shades of gray in reflected light. The thickness of each of the 12 sections was determined shortly after they were placed on the grid. These determinations were made by two different observers 10 times per section to allow for calculation of errors. The same sections were exposed to a vacuum of 2 × 10 -~ mm Hg for 60 minutes, whereupon their thickness was measured once again. They were next introduced into an electron microscope (Akashi Tronscope) and subjected to irradiation for 5 minutes at an intensity such that focusing was possible at a primary magnification of x 15,000. Then the thickness of the sections was measured a third time. The second series of thickness determinations was made on serial sections cut on the Ultrotome at two different microtome feeding rates, one at 250 A and another at 100 A per section. The length of the uninterrupted series varied from 20 to 60 sections. The purpose of these determinations was to find the amount of distortion of the sections from compression at the cutting. Serial sectioning was necessary to avoid inadvertently measuring sections cut at a multiple of the feeding rate due to missing of the foregoing cut.

RESULTS The e r r o r of a thickness d e t e r m i n a t i o n of a section was calculated as the t o t a l error, 2

e = ] / ~ + era, where e~ is the i n d i v i d u a l error a n d 8m the m e a n error, i.e., the difference

between the m e a n thickness as d e t e r m i n e d b y two observers. The t o t a l e r r o r has been calculated to be ~< 10 % at the thickness range of 300-700 A. N o statistically significant difference was f o u n d between the observations of the two observers (Table I). The m e a n thickness of the first series of u n t r e a t e d sections is 472 A ; the thickest section is 740 A a n d the thinnest 340 A. A f t e r v a c u u m t r e a t m e n t the m e a n thickness of the same sections decreased to 425 A, a difference of - 47 A. H o w e v e r , the s t a n d a r d d e v i a t i o n of the differences is large (72 A), which m a y be i n t e r p r e t e d as due to variability of the i n d i v i d u a l sections u n d e r v a c u u m treatment. E x p o s u r e of the sections to the electron b e a m r e d u c e d their m e a n thickness to 208 A, a difference of - 264 A. Changes in thickness, expressed as a percentage of the u n t r e a t e d value, r a n g e d between - 4 0 % to + 30% for v a c u u m t r e a t m e n t a n d - 7 5 % to - 4 0 % for b e a m t r e a t m e n t , estimated at an u n c e r t a i n t y of 10%. The fairly large figure of u n c e r t a i n t y reflects the small n u m b e r of sections measured. 1 Manufactured by AB LKB-Produkter, Stockholm, Sweden. Mikrokator 509-9, C. E. Johansson, Eskilstuna, Sweden.

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T. ZELANDER AND R. EKHOLM

In the second series of d e t e r m i n a t i o n s the m e a n thickness of serial sections was 326 A and 493 A at an average m i c r o t o m e ad v an ce of 100 A and 250 A respectively (Table lI). Th e difference between the m e a n thicknesses is statistically significant on the 1% level. The thicknesses of the single sections in the 100 A series is given in Tab l e III. T A B LE I M E A N THICKNESSES A N D S T A N D A R D DEVIATIONS IN fik OF

10

DETERMINATIONS OF

12

AFTER S E C T I O N I N G A N D TREATMENT I N V A C U U M A N D W I T H ELECTRON BEAM Diff.

Diff.

Untreated

Vacuum treated

/~

%

Beam treated

/~.

%

s~

472 126

425 106

-47 72

-9 15

208 60

-264 117

-59 8

~+ 2s~

724

637

328

TABLE II AVERAGE MICROTOMEFEEDINGSAND SECTIONTHICKNESSESIN Diff. = 167. taifr" = 2.93 (significant difference). Feed

100 ~

Mean s2 n

250

326 9390 10

493 17,262 6

TABLE III T H E THICKNESSES OF T H E SECTIONS I N T H E 1 0 0 /~k SERIES Section no.

Thickness 2~

1 2 3 4 5 6 7 8 9 10

463 463 227 276 364 339 409 255 190 276

SECTIONS

T H I C K N E S S OF E L E C T R O N M I C R O S C O P Y SECTIONS

417

DISCUSSION The accuracy of the measuring method described has been determined to be 10 (4). Less accurate determinations may result under less favorable conditions, such as folds close to the measured area of the section. In the present investigation the total error is calculated to be ~< 10% for the thickness range of 300-700 A. To determine the original thickness of methacrylate sections prior to their insertion in the electron microscope it appears necessary to avoid methods which include vacuum or electron beam treatment. Electron bombardment, particularly, results in a marked loss of section material (mean value: 60%), which has also been pointed out previously (9, 16). But vacuum treatment alone may change the section thickness. Since the geometrical thickness (t) of the section is calculated from the optical path difference of the light going through the section and passing by it, a change in the refractive index of the methacrylate resulting from vacuum or electron beam treatment will change the calculated thickness value. Therefore, by use of the interference microscope, both the geometrical thickness and the refractive index of each of 10 sections, ranging from 1 to 3 # in thickness, were determined before and after vacuum treatment by immersion in two media with known refractive indices (2). The thicknesses were checked by an independent method (3). The results revealed no statistically significant difference in refractive index before and after vacuum treatment because of the large standard deviation. It seems possible that adsorption of small quantities of contaminants to the surfaces of the sections or evaporation of volatile impurities from the methacrylate could be the cause of this variability. The thickness values of sections exposed to the electron beam could be questioned since they were calculated using the refractive index of untreated methacrylate. However, as the optical path difference of these thin sections is only about 10 times the accuracy limit of the interference microscope, the calculation of their refractive indices based on immersion in different media gives only very rough values. The indices of thicker sections have been determined, but the figures are of doubtful usefulness as the action of the electron beam on these sections is certainly more intense due to the proportionally higher heating of the sections. However, the decrease (approximately 60%) in thickness following exposure to the electron beam, as measured with the interference microscope, is reasonably close to Reimer's (9) figures (up to 55 %) based on an electron transmission measuring method. The thicknesses of serial sections were considerably higher than the microtome feeding rate, especially for the 100 A series. As the measurements were made on uninterrupted serial sections this difference was interpreted as mainly due to compression of the sections. This is compatible with Peachey's studies (5, 6), in which it was

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T. Z E L N A D E R A N D R. E K H O L M

proved, by measuring the length of the sections in the direction parallel to the direction of cutting, that there is considerable compression, even of much thicker sections than those used here. He found, for example, that a section showing gold interference color when first cut is usually reduced in length to about 60 % of the corresponding dimension of the block (5). A certain deformation due to compression is always found in sections of blocks containing biological material. By sectioning small particles arranged in a regular three-dimensional pattern it can be proved, first, that the layer of material removed from the specimen has a thickness fairly well corresponding to the feeding rate of the microtome and, second, that compression of the section gives rise to a closer packing of the particles in the direction of sectioning (15). Similar conclusions can be drawn from the deformed pictures of structures known to be spherical. However, the compression generally observed in electron micrographs of biological material does not seem to be as pronounced as that found in this study. This is obviously due to the fact that the embedded material reinforces the block. For some biological tissues this reinforcement seems to be very strong, resulting in a compression of the specimen of only about 10%, as shown by Sj6strand (14) in myelin sheath. Some commonly used manipulations, such as stretching the sections on the liquid surface with xylene vapor, also tend to neutralize the compression effect of sectioning. It is to be noted, too, that embedding materials other than methacrylate, e.g., Araldite, seem to suffer less compression on sectioning. Sj6strand (13), using a shadow-casting method, studied the thicknesses of serial sections of about the same thickness range as the 100 A series studied here. He obtained a mean thickness value (237 A) which was somewhat lower than that of the present authors'. The main reason for this discrepancy seems to be that Sj6strand measured the thickness of the edges of sections or fragments of sections. Other factors that probably tended to reduce his values are the vacuum treatment and, as he pointed out, deposition of material from the liquid surface onto the supporting film prior to shadow-casting. However, Sj6strand did not claim that the values obtained represented the absolute thicknesses of the sections, but recommended his procedure as a simple and useful standard method for estimating the relative thicknesses of the sections obtained in different laboratories and with different microtomes. The thickness of sections showing the desired gray color in reflected light was found by S6chaud et al., making use of phage particles, to be about 400/~. These authors claim that most sections (of Vestopal W) used in their laboratory probably fall within the range of 300-600 A. That this is the most used thickness range seems to be the general opinion among electron microscopists. In the present study a microtome advance of 100/~ per section gave a series of sections with mean thickness

THICKNESS OF ELECTRONMICROSCOPYSECTIONS

419

value of 326 A, the thinnest sections being about 200 A. Since the blocks used did not contain any tissues, and since no special efforts were made to remove the sectioning distortion, these thickness values undoubtedly are considerably higher than those usua!!y resulting from sectioning biological material. When aiming at high-resolution electron microscopy, it is evidently possible to produce sections appreciably thinner than those commonly claimed to be in use, a fact previously emphasized by Sj/Sstrand (12). ADDENDUM Since the completion of this study, A. Cosslett has presented a paper at the European Regional Conference on Electron Microscopy at Delft, 1960, on the effect of an electron beam on thin sections. Cosslett has determined the thickness and the refractive indices of different embedding media before, and after the electron bombardment, with the aid of the light interference technique. As discussed above, it is impossible to determine interferometrically, with enough accuracy, both the thicknesses and the refractive indices of sections within the thickness range common in electron microscopy. Cosslett has avoided this difficulty by using sections of 1500-2000 ~ in thickness. The action of the electron beam on these sections must be considerably more intense than on sections of the thickness studied in the present investigation.

REFERENCES 1. BACHMANN,L. and SITTE,P., Proc. lntern. Conf. Electron Microscopy, Berlin, 1958, p. 75. 2. DAVIES,H. G., in DANTELLI,J. F. (Ed.), General Cytochemical Methods, p. 116. Academic Press, New York, 1958. 3. HALL~N,O., Acta Anat. Suppl. 25 (1955). 4. JOHANSSON,L. P., Exptl. Cell Research Suppl. 4, 158 (1957). 5. PSACH~Y,L. D., J. Biophys. Biochem. Cytol. 4, 233 (1958). 6. - - - - Proc. Intern. Conf. Electron Microscopy, Berlin, 1958, p. 72. 7. PORTER,K. and BLUM, J., Anat. Record 117, 685 (1953). 8. REIMER,L., Naturwissenschaften 44, 335 (1957). 9. - Electronenmikroskopische Untersuchungs- und Pr~iparations-methoden, Springer Verlag, Berlin, 1959. 10. S~CHAUD,J., RYTER,A. and KELLENBERGER,E., J. Biophys. Biochem. Cytol. 5, 469 (1959). 11. SJ6STRAND,F. S., Experientia 9, 114 (1953). 12. - Z. wiss. Mikroskop. 62, 65 (1954). 13. - First European Regional Conference of Electron Microscopy, Stockholm, 1956. 14. - in CUMINGS,J. N. (Ed.), Modern Scientific Aspects of Neurology, p. 188. Edward Arnold Ltd., London, 1960. 15. SJ0STRAND,F. S. and POLSON,A., J. Ultrastructure Research 1, 365 (1958). 16. WILLrnMS,R. C. and KALLMAN,F., J. Biophys. Biochem. Cytol. 1, 301 (1955). 17. ZELAND~R,T. and Er~I4OLM, R., Proc. lntern. Conf. Electron Microscopy, Berlin, 1958, p. 84.