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PARAEFIN COMPRESSION DUE TO THE ROTARY MICROTOME
WILFRID TAYLOR DEMPSTER, Department of Anatomy, University of Michigan, Ann Arbor, Mich.
ABsTmcT.-The extent of compression of microtome sections has been studied for blocks with tissue and also blocks of clear pa€6n. Thick sections are commonly compressed 15% or more, while in sections below 5 or 10 p, compression may exceed 50%. Compen- satory thickening of sections occurs. The degree of compression for various p a r f f i samples and for various conditions of knife edge, temperature, etc., is compared. Microscopical work, particularly where quantitative data or reconstructions are involved, is often seriously impaired by unrecognized artifacts of sectioning. The present work indicates the magnitude of such artifacts. Com- pensation for distortions of sections is not easy because tissues, particularly dense tissues, may compress less than the paraf61.1 matrix. Section corrugation is due to this inequality in compres- sion. Absorption of water in section flattening causes some tissue readjustment, but this varies with different tissues and different kations.
Distortions of tissue by the microtome knife are commonly recog- nized, but the extent and nature of such defects have not received sufficient attention to aid in their critical evaluation. A study of the distortions induced by the microtome properly involves: (1) a deter- mination of the amount of shortening of paraffin ribbons occurring under conditions common in routine sectioning; and (4) the altera- tion of conditioning factors, one by one, in relation to controls, to determine which factors increase and which decrease the distortion. Such a study is approached most effectively by experiments involving homogeneous blocks of paraffin without included tissue. Thereafter, the modifications due to tissue may be examined.
THE EXTENT OF PARAFFIN COMPRESSION The technic of determining the degree of compression of ribbons
from the rotary microtome was simply the measurement of lengths of ribbon and the computation of section length to block length ratios (i-e., compressed length ratio) and of the percentage of compression. Routinely, to form one group of data, such ratios were found for seven or eight representative thicknesses of section within the range 1-25 p.
SAIN TECHNOUIQY. VOL. 18, No. 1, JANUARY. IS43
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19 STAIN TECHNOLOGY
To obtain compression data reflecting the degree of compression found in routine sectioning, homogeneous blocks of 50-52' melting point (m.p.) paraffin (Bioloid paraffin of Will Cop., Rochester, N. Y.) were sectioned by Spencer and Leitz microtomes. The freshly melted paraffin was poured into paper boxes and cooled in water of 18-20' C. Blocks were trimmed to cutting face dimensions of 10 mm. In order to insure that blocks were the same size and had parallel sides, the jaws of a vernier caliper were forced over the blocks after they were nearly trimmed to size. The blocks were sectioned a t room temperatures ranging from 20-26' C., and a sharp nick-free knife having a 27' facet bevel was used. Clearance angles' between the inner facet of the knife and the block face (see Fig. 1) ranged from 1-6'. Compression data based on the above somewhat variable conditions should be a fair approximation of the degree of cornpres-
TABLE I. DISTORTED DIMENSIONS OF PARAFFIN SEC~~ONS DUE TO TEE ROTARY MICROTOME WITH COMPUTAT~ONS OF COMPENSATING SECTION TAICKNEBS.
Percentage compression
Micmtome setting (micra)
53.0 20.0 15.0 14.5 10.0 7.5 5.0 4.5
Computed thickness (micra)
ection length X lo( Block length
20.2 20.3 21.9 53.3 a7.5 40.9 57.8 83.9
79.8f4.5 79.7*4.5 78.1f4.6 74.7f5.5 72.5f6.7 59.8f6.9 44.2f8.1 16.1zt6.9
31.2 45.1 19.2 16.7 13.8 12.6 11.9 15.5
sion obtained in routine sectioning where sectioning temperatures and rake have not been rigorously controlled.
The average magnitude of compression distortion, based on 40 or more determinations .for each thickness of sectioning, is represented in Table 1. The ratios pertain to ribbons flattened by floating on water of about 40' C. Compressed ribbons are shortened on the average by 20% or more. Below lop , sections show increasingly severe compression, and very thin sections may be only a fifth of block length.2 Likewise, below l o p , the variability of compression increases.
'The bevel angle of a knife plus the clearance angle used in cutting represent the amount of tilt of the outer or forward facet of the knife. Commonly, and this defini- tion will be followed henceforward, the combined angle is defined in terms of rake angle; rake angle=W - (bevel angle+cleamnce angle).
'The Weo curve of Fig. !Z represents a graphical presentation of similar data, in which. however, the paraffin was cut at a temperature of 26.5OC. and a knife rake of 70°.
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PARAFFIN COMPRESSION 15
Paraffin ribbon width was not appreciably different from block width. Compensation for section shortening was effected by an increment in thickness. The expected average thickness of sections, assuming the paraffin volume to be unchanged, has been tabulated. Paradoxically, ribbons made with a very thin microtome setting were sometimes actually thicker, due t o compression, than those made at a thicker setting. Measurements of section thickness, made with a calibrated microscope fine adjustment, a relatively coarse method of measurement, were invariably more than enough to compensate for section length. Richards (1942) has measured this “excess thick- ness’’ by more elaborate methods; excess thickness is doubtless due to stress opacity (Dempster, 1942a) and associated density changes.
In sectioning with the oblique knife of the sliding microtome, thin sections are likewise more compressed than thicker ones, and a transi-
’
FIG. 1. Diagram representing a microtome knife in contact with a block of paraffin. Pertinent angles are indicated.
tion similar to that with the rotary microtome occurs at about 10 p (Dempster, 1949b). In addition, warping defects are produced by the sliding microtome.
CONDITIONING FACTORS In order to determine which factors increased and which de-
creased compression, experiments were performed in which specific conditions were varied in relation to controls. The conditions ordinarily used as a standard were: 97’ knife bevel, band sharpened acute edge, 1’ clearance angle, freshly melted 5%5P C. m.p. Bioloid paraffin, blocks cooled in water of 18-20’ C. and trimmed to 1 X 1 em., and cutting speed of one crank turn per second (i.e., 30.9 ft. per minute).
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16 STAIN TECHNOLOGY
Increased cutting speed, 1.5 and 2 crank turns per second, made no significant differences in average compression. A knife with a rough and jagged edge induced uneven and more than usual compression as compared with a smooth sharp edge. Likewise, a clean sharp edge dulled smoothly by rubbing a hone along it caused noticeably com- pressed sections for each thickness, and this effect was particularly marked for thin sections. In Richards' study (1942) of knife sharp- ness, edges sharpened by the finest abrasives and by careful stropping produced less section compression than knives sharpened with coarser abrasives.
The angIe of knife rake had a very Significant effect on compres- sion. As the advance facet of a knife was flattened from a position
RAKE
L ! j , i , I
FACET TILT
I 1
0" zoo 30° 40° 56 60- 700 86 96 106
Fro. %. The relative effect of kniie rake angle on the extent of compression of paraffin sections for five thickness settings of the microtome.
perpendicular to the block face, i.e., from a 0" rake, sections of any thickness became noticeably less compressed. Fig. 2 shows this for samples of 56-58" C. m.p. Bioloid paraffin sectioned at a temperature of 30°C. Several knives were used in sectioning including one spe- cially honed to a 17" bevel angle, which permitted unusually wide rake angles. In addition t o showing smaller compression for wide rake angles, the figure shows that, as the rake is increased beyond about SO", compression is more than proportionately reduced. By way of contrast, scraping cuts produced by low order and negative rakes caused augmented compression. Thinner sections showed less rela-
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PARAFFIN COMPRESSION 17
tive compression with increased knife rake than thicker ones. From this, it will be evident that wider rakes than are now common (i.e., 55-60') may be useful in practical sectioning. It is at least desirable to know the bevel angles of available knives, and it may be ad- vantageous to prepare more acutely beveled knives than are com- mercially available. Von Ardenne (1939) on the basis of a theo- retical analysis has likewise come to the conclusion that acute knife bevels and low clearance angles are required for minimal distortion of sections.
Little difference in compression was evident when paraffin blocks were cooled in ice water or were allowed to cool more slowly by ex- posure to air a t room temperature. Actual sectioning, of course, was done after blocks were at the same room temperature thruout. Some blocks were cooled from the bottom of the embedding box while the
6 0
m > 40 0
_I
0 5 10 15 20 25
SECTION THICKNESS IN MlCRA FIG. 3. Compression curves for paraffins of different melting paints. Paraflin melt-
Sample 45O is from Grtibler; others are Will
surface was kept free of congealing paraffin by playing a hot spatula over it. This resulted in a predominantly vertical orientation of the crystal axes of the paraffin. Significant difference in compression did not appear between sections which were cut across and those cut along the length of the principal axes of the crystalline pattern. In some blocks, often those cooled very rapidly, tiny but visible air spaces appeared, and, when such blocks were sectioned, compression was greater than in similar paraffin without such spaces. Blocks of paraffin of different sizes (i.e., cutting faces measuring 5x5 mm., 5x10 mm., 5 x 9 0 mm., and 10x90 mm.) were found to compress to essentially the same degree as the standard 10x10 mm. block.
ing points are indicated by figures. Corp. "Bioloid" para5ns.
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18 ST.4IN TECHNOLOGY
Paraffins of different melting points were sectioned a t a rake of yoo and at a rmm temperature of 26.5"C., and the compression curves were in general of similar shape (Fig. 3). The curves show a rough, but inexact, correlation with the paraffin melting points. Lower melting point paraffins showed much more compression than harder puaffins. The section thickness a t which a marked degree of com- pression began was less for harder than for softer paraffins. Like- wise, the possibility of cutting usable sections of thickness less than 5 p became greater with harder paraffins.
Aumonier (19S8), after measurements of paraffin compression, pointed out that a 52" C. m.p. paraffin showed greater compression &an a 58°C. m.p. paraffin. This and the data of Fig. 2 obviously suggest that melting point-a temperature at which the main mass of a paraffin is liquid and a small proportion is in the solid s t a t e i s an inadequate criterion for properties (sectioning behavior) char- Wteristic of the solid state. A better index of the cutting properties of paraffin would seem to be the plastic point-a transition point in the plastic properties of solid paraffin. (See Dempster, 1942a,). The compression curves of Fig. 3 fall into three groups: two curves in- dicating great compression, three with slight compression, and an intermediate curve; the plastic transition points of the same paraffins fall into similar groups.
Much confusion appears in attempts to compare the sectioning qualities of paraffins, since two factors are involved, compression and ease in obtaining ribbons. For section thicknesses to the right of the heavy line (X) in the figure, ribboning was dificult or impossible at the cutting temperature used. When the room temperature was raised, ribboning of the thicker sections became possible, and, if the temperature was raised enough, sections much thicker than 25 1.1 could be ribboned easily. The line (X), representing the transition between ribbon formation and discontinuous sections, in effect moved to the right. The degree of compression at warmer temperatures simultaneously became greater, and this compression was marked for thin sections and for low melting point paraffins.
When one of the harder paraffins (56-58OC. m.p. Bioloid) was sectioned in a warm room a t a temperature of 35OC., the compression curve was like the two lower curves of Fig. 2, but compression was slightly more marked. Ribbons of 25 p or greater were easily ob- tained. Sectioning, however, when the temperature of the block, knife and room was reduced to 15OC., resulted in a curve that was in no way different from that 6gured for the same paraffin when cut at 26.5T. (Fig. 3). On the other hand, the softer 47-49OC. m.p.
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PARAFFIN COMPRESSION 19
paraffin showed less compression when sectioned in a cold room at 9°C.; a curve similar to that figured (Fig. 3) for 50-52OC. m.p. paraffin was produced. Paraffin with a 5&5e°C. melting point sectioned a t 9°C. showed some slight reduction of compression when compared to the compression values obtained at a normal room temperature. It would thus appear that marked reduction of sectioning temperature has a distinct effect in reducing compression for low melting point paraffins, but little effect on hard paraffin, and a moderate effect on intermediate paraffin.
In general, the transition thickness, below which compression be- comes increasingly marked, acquires lower values as the temperature of sectioning is reduced and higher values as the temperature of cutting is raised. Thus, if the transition thickness of a given paraffin as indicated by a compression curve is about 10 p for ordinary room temperature, the transition thickness may become 5 p or less with a suitably adjusted cold temperature or 15 I.( or more with warm sec- tioning. Tho cold sectioning may produce less distortion of thin sections, the possibility of obtaining ribbons of thicker sections, in- stead of discontinuous curled sections, decreases with cold.
Optimal sectioning may be defined as the smallest possible degree of distortion compatible with the obtaining of ribbons. Optimal sectioning a t 45 p is represented in Fig. 3 by the 50-52”C. m.p. paraffin a t the temperature tested, or at some lower temperature a t which ribboning is still possible, or by the harder paraffins at a suit- ably adjusted higher temperature. For the harder paraffins 15 p sections are best at the testing temperature (26.5”C.) or a t lower temperatures which still permit ribboning. Very thin sections ap- parently cannot be produced with the same minimal degree of com- pression as thicker sections, tho hard paraffin sectioned at ordinary or reduced temperatures is better than soft for moderately thin microtome settings. Practical means of reducing compression for thin sections to still smaller values are increased knife rake, obtained with acutely beveled knives, and methods of celloidin double em- bedding or both. Possibly, wax adulterants may be found which will produce an embedding mass not characterized by the downward inflection of compression curves for thin sections.
Galigher (1934) has provided a rough practical guide of cutting temperatures to be used with two types of paraffin when sectioned at various thicknesses. Presumably similar recommendations based on “optimal sectioning”. as defined, could be prepared. Waterman (1939), Hance (1940) and Groat (1941) have used parafin adulterants to increase the hardness of embedding matrices. It would be a dis-
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eo STAIN TECHNOLOGY
tinct advance if embedding waxes could be prepared so that when sectioned under standard conditions-temperature, knife rake, knife sharpness, etc.-"optimal sectioning" would result, and compression for sections of predetermined thickness would be less than about 10 or 15%. Methods of standardization applied to the physical proper- ties of the solid state of waxes (plastic point, etc.), in addition to the melting point, are a necessary prerequisite. This standardization, by methods now available, becomes particularly desirable as paraffins containing adulterants come into increasing use.
SECTION FLATTENING When sections are floated on water and warmed above the plastic
transition point, but below the melting range, the paraffin softens appreciably. Because of the viscous nature of paraffin in this thermal range, surface tension forces eliminate the natural curling of sections and the ripples due to partial uncurling a t room temperature. If the stretching temperature is about ten degrees below the melting point, good expansion due to surface forces occurs. Ribbons, however, are very soft and if the slide on which sections rest is held upright, the ribbons will elongate and become narrow.
With favorable conditions of stretching, expansion of sections from the rotary microtome amounts to 6% of original block length for 5 p seetions and regularly decreases to 2.3% for 25 I.L sections (56-5S°C. m.p. p a r d n ) . Thus in the curves figured, if ribbons had not been flattened, distortions would have been slightly more than indicated. If one ignores cases in which thinner sections may not flatten com- pletely, expansion for complete flattening rately differs by as much as 1% from the figures indicated. This applies equally well to sections made thruout a range of knife rake from 45O to 70'.
n 5 5 U E COMPRESSION A group of measurements was made on ribbons from blocks
(50-52"C. m.p. paraffin) containing embedded tissue (liver, muscle and embryo), and it was found that compression curves for whole sections were almost identical with that found for paraffin alone. The blocks to be sectioned each contained a cylinder of tissue which had been turned on a lathe from a previously embedded mass of tissue. Cylinders were prepared so that their cross-sectional area amounted to half (actually 47 to 52%) of the block face. Measure- ment of the compressed ellipses of tissue in the sections after flatten- ing showed that the ratio of the short to long axes for each section thickness was equivalent to the compression ratios of whole sections
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PARAFFIN COMPRESSION 21
of similar thickness. The same is true if a cylinder of paraflin- infiltrated celloidin (4%) is re-embedded in paraffin and sectioned with the rotary microtome.
Differences between tissue and celloidin ellipses, however, make untenable the obvious conclusion that tissue and matrix compress to the same degree. Two modifying factors require consideration.
In sections containing celloidin, even after a p a r f i n ribbon has been warmed and stretched to its maximum, the surface of the celloidin portion of the section is rippled with wavy folds having long axes transverse to the axis of compression. The surrounding paraf- fin, however, is flat and free from ripples. Thus, it appears that celloidin is actually compressed less by sectioning but is held in a frame too narrow. Compensatory corrugations result.
If the celloidin ellipses are freed by teasing or by cutting away the surrounding paraffin, they may be flattened free of undulations by floating on warmed water. Under these conditions, the true com- pression varies from 4.5% for 25 p sections to 16.4% for 10 p sections and to 20.2% for 5 p sections. This is obviously less compression than is indicated in Table 1 and serves to explain the great folding of celloidin, particularly in the thinner sections. When tissue instead of celloidin is sectioned, i t likewise becomes corrugated; the amount of folding, however, varies from tissue to tissue. Denser tissues com- press less than paraffin and show more folding.
The second modifying factor is evident with tissues but not with celloidin. When tissues are floated on warm water, the rippled tissue tends to flatten. bfter flattening, the thicker sections, at least, are free, or largely free, from wrinkling. Tissue slices tend to adjust their surface area to that allowed by the surrounding frame of paraffin. Moreover, when freed tissue slices are floated on water, they expand laterally as well as along the section length. This effect occurs likewise if the slices are flattened on warm alcohol. The area occupied by such a section is appreciably greater than that covered by a similar piece of dry tissue from a section flattened me- chanically by brush tips. When the peripheral frame of paraffin surrounding a tissue slice is trimmed so that only a very narrow band of matrix is present, tissue expansion may distend the frame; this is particularly true with thin sections. I t appears that both the adjust- ment of a tissue slice to its paraffin frame and the expansion of the unenclosed tissue are accompanied by the absorption of water, soften- ing of the tissue and tissue swelling. The softening of embedded tissue on immersion in water has been recognized by Chamberlain (192o), Galigher (1934) and Johannsen (1955) and recommended as a
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PO STAIN TECHNOLOGY
procedure for making possible the sectioning of refractory tissues. A further indication of water penetration into paraffin infiltrated tissue was made by Copeland (1941) who demonstrated that aqueous dyes penetrated and stained embedded tissue.
Ko data on differential adjustment of tissues thru water absorption nor information on the relative degree of compression of the same tissue fixed in various ways are at hand. The physico-chemical properties (swelling, stainability, hardness, for instance) of various tissues differ following the various types of fixation; accordingly, differences may be expected in both of the factors under discussion. Differences in water absorption, as noted above, are marked between tissue and celloidin. Appreciable differences, likewise, are apparent in the relative absorption of water by tissues depending on fixation. Freed tissue slices from ribbons of lathe-turned and re-embedded cylinders of liver showed noticeable expansion of the compressed ellipses if the tissue had been preserved in 10% formalin. But, with preservation in saturated sublimate or saturated picric acid, there was much less expansion.
CONCLUSIONS
Section compression to some degree is an inherent consequence of the mechanics of sectioning, but the extent of such compression de- pends on several variable factors. Conditions for minimal compres- sion are as follows:
1. The knife should be keen and even along the span used in cutting.
$2. The honed facets should be smooth and as free of scorings as possible.
3. The rake angle should be as wide as possible. The clearance angle should be positive but small. Measurement of these angles is recommended. Special honing of narrower than usual bevel angles may be of value.
4. Gummy accumulations of paraffin including that on the inner facet should be wiped away.
5. The paraffin surrounding a tissue should be clear and homo- genous-without mottling, opacity or air spaces.
6. Cutting temperatures should be adjusted to the hardness of the paraffin used, and in general, the harder (i.e., high plastic point) paraffins are desirable.
7. The cutting temperature should be just warm enough, in rela- tion to the plasticity and hardness of the paraffin, to permit ribboning. Marked cooling will be required for thinner sections. ,
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PARAFFIN COMPRESSION 2s
Curves of paraffin compression, based on ribbons of different thick- ness, shows a deflection indicating much greater compression for thin sections than for thicker ones. Since some compression is unavoid- able, since the magnitude of compression may be great, and since compression varies with section thickness, measurements (linear, surface, volumetric) made upon microscopical material must a priori be regarded as untrustworthy unless the extent of compression is evaluated. Errors in plastic and graphic reconstructions and even in qualitative work as well may be introduced if compression is severe. Whenever one sections material that is to be used for meas- urement or critical observation, the extent of compression should be determined for the specific conditions of sectioning.
The degree of paraffin compression characteristic of a paraffin matrix provides a maximum compression value which tissue com- pression may approximate. A tissue may compress as much as or less than its paraffin matrix. If tissue compression is less than that of the paraffin matrix, corrugation of the tissue area results. This defect becomes less or is eliminated if the hardness of the matrix is increased or if the tissue hardness is decreased. The practice of infiltrating in a soft paraffin and embedding in a hard one is helpful in this respect. On flattening, the tissue absorbs water, softens, and adjusts its area to that of the frame of paraffin a t the periphery of the section. If tissue area is noticeably greater than the area of the frame of paraffin, corrugations will persist. The effectiveness of adjustment during flattening varies with tissues and their fixation.
REFERENCES
TON ARDENNE, M. 1939. Die Keilschnittmethode, ein Weg zur Herstellung von Mikrotomschnitten mit weniger als 10- mm. StLrke far elektronen mikroskopische Za-ecke.
AUMONIER, F. J. 1958. Notes on the distortion of paraffin sections. J. Roy. Micr.
CHAMBERLAIN, C. J. 1990. Methods in plant histology. rniv. of Chicago Press.
COPEIAND, D. E. 1941. The surface staining of tissues embedded in paraffin.
DEMPSTER, W. T. 194%. The mechanics of paraffin sectioning by the microtome.
. 1949b. Distortions due t o the sliding microtome. Anat. Rec., 84,
1934. The essentials of practical micmtechnique in animal biology.
New paraffin-resin infiltrating and embedding media for micro-
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Anat. Rec., 84,44147.
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GROAT, R. A. A. E. Galigher, Inc., Berkeley, Calif.
technique. Science. 93, 311-2. 1941.
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94 STAIN TECHNOLOGY
HANCE, R. T.
JOEANNSEN, D. A. 1935. Dehydration and infiltration. Science. 82,253-4. RICHARDS, 0. W. 1945!: The effective use and care of the microtome. Spencer Lens
WATERMAN, H. C. The preparation of hardened embedding para511 having
1940. The flow point and not the melting point is important in his- tological waxes. Anat. &., 78 (Suppl.). 118.
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low melting points. Stain Tech.. 14, 55-69. 1939.
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