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Artifacts observed in critical-point-dried preparations of human chromosomes by electron microscopy
JOURNAL OF ULTRASTRUCTURE RESEARCH 73, 27--33 (1980)
Artifacts Observed in Critical-Point-Dried Preparations of Human Chromosomes by Electron Microscopy 1 G. F. B A H R A N D W. F. E N G L E R Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306 Received May 21, 1979, and in revised form June 25, 1980 Preparations of eucaryotic c h r o m o s o m e s tend to suffer from the effects of moisture in criticalpoint drying and from mechanical stretching during preparation, resulting in fusion of fibers so that the latter are ultimately transformed into thick strands. Mechanical stretching alone is accompanied by s o m e fusion, but it m a y render insight into the architecture of c h r o m o s o m e s .
200 A in average diameter (1--6). These fibers are v e r y easily stretched and fuse readily with their neighbors; furthermore, they can be damaged during preparation by harsh fixation, dehydration, extractir/g milieu, or mechanical forces. In this article we will describe some damaged chromatin fi~ bers, as we have observed them.
The analysis of the architecture of chromosomes is currently pursued in many laboratories. Some use, as we do, the method of critical-point drying. A relatively new method of preservation or fixation applied to a biological object about which relatively little is known, such as the fine structure of the mammalian c h r o m o s o m e , might present crucial problems as to the " r e a l i t y " of the o b s e r v e d object. In the case of the mammalian chromosome, a definitive answer as to reality is made more difficult because the structural details (e.g., chromosomal constrictions and banding features) are either clearly within the limit of resolution of light microscopy or they are clearly b e y o n d its resolution, as fibers with a diameter of 200 A would be. The observation of fibers with such a dia m e t e r as the principal feature of their structure, therefore, is obviously in need of repeated confirmation. Also needed are diverse preparatory approaches and multiple observations using the same preparatory method to attest to the invariability of the fiber appearance. There is now general consensus that the fiber of chromatin in the nucleus and in chromosomes of humans is a knotty, contorted construction of about
M A T E R I A L S AND M E T H O D S H u m a n cells from tissue culture were h a r v e s t e d after a 2- to 4-hr exposure to colcemid for mitotic arrest. They were then e x p o s e d to h y p o t o n i c H a n k s ' balanced salt solution, 1:1 dilution, or to distilled water, both of which induced the cells to swell. U p o n centrifugation, s o m e of the swollen cells were placed onto the surface of water in a L a n g m u i r - t y p e trough. T h e y were picked up on carbon-coated F o r m v a r grids and critical-point-dried f r o m e t h a n o l or a c e t o n e . U n stained c h r o m a t i n was viewed in the electron microscope at 100-kV acceleration of voltage. RESULTS
The distinguishing features of criticalpoint-dried chromatin fibers considered as well preserved are (a) a distinct, sharp cont o u r in t w o - d i m e n s i o n a l images, (b) a rounded, although irregular, shape when viewed in stereopairs, (c) a relatively constant diameter at various sites in the nucleus or chromosome, and (d) a relatively uniform density (electron scattering property) along the length of the fiber. When two or more fibers cross, the electron scattering power at the crossover point is doubled or multiplied in a predictable, measurable
The opinions or assertions contained herein are the private views of the a u t h o r s and are not to be c o n s t r u e d as official or as reflecting the views of the D e p a r t m e n t o f the A r m y or the D e p a r t m e n t o f Defense. 27
0022-5320/80/100027-07502.00/0 Copyright O 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
28
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FIG. 1. A limited area out of a m e t a p h a s e illustrating s o m e recognizable but not well-preserved chromosomes. In the lower left corner can be seen a totally clumped c h r o m o s o m e , w h o s e fibers merge with the rest of the c h r o m o s o m a l material in the middle o f this field. N u m e r o u s particles of approximately spherical shape m a r k the e x t e n s i o n of loops and naked D N A (not visualized without staining or s h a d o w casting) into the background w h e r e they adhere to the carrier protein film essential to this type of preparation. The preservation of the c h r o m a t i n is generally severely affected. × 62 000. Inset: View of well-preserved nuclear chromatin fibers, × 100 000, A F I P neg. 78-7011-1.
A R T I F A C T S IN C H R O M O S O M E S
manner. Similarly, the transradiated mass increases when the fiber is folded back on itself or runs a course parallel to the direction of observation (on a positive print the contrast of the fiber will be seen to be increased). Features a, c, and d are illustrated in insets to Fig. l (nucleus) and Fig. 2 (chromosome). It is our experience from working with critical-point-dried c h r o m o s o m e s for the last 10 years that the normal appearance of the chromatin fiber, e.g., its diameter, is narrowly distributed (insets to Figs. 1 and 2). Foremost among the alterations fibers undergo are stretching and fusion. In the worst of instances both occur at the same time, and a c h r o m o s o m e may be so clumped that its identity as a chromosome is actually in question. Clumped and coalesced fibers are readily recognized in Figs. 1 and 2. Some affected chromosomes are better preserved than others, although riot opti-
29
really, but there can be no question as to whether they are chromosomes. It is more difficult to r e c o g n i z e the e x t e n d e d , smudged, centromeric area in the chromosome shown in Fig. 3 as an artifact, because the rest of this c h r o m o s o m e is relatively well preserved. The pattern of more or less well-preserved fiber loops emerging at the sides of chromatids documents the relatively high level o f p r e s e r v a t i o n . T h e r e is n e v e r t h e l e s s a s t r a n d o f c l u m p e d and stretched chromatin in the upper left corner of Fig. 3, signaling that this preparation contains artifacts. Only one or two fibers (arrows) retain their natural shape and proper diameter of 200 ]~. Fusion or coalescence of chromatin fibers is, as far as we can determine, chiefly due to faulty critical-point drying of the unfixed chromatin. Either incomplete removal of water from or an excess of moisture in the dried preparation causes the obliteration of individual fibers.
FIG. 2. Partial m e t a p h a s e plate. T h e c h r o m o s o m e s hang together. T h e fibrous nature is d e a r l y recognizable in m o s t of them. In the middle and lower right, c h r o m o s o m a l structure is severely affected by fiber clumping and fusion. T h e crispness of the individual fiber is lost; only a few fibers above the carrier film retain their original d i m e n s i o n s and individuality (arrows). × 10 800. Inset: View of well-preserved telomeres of a chrom o s o m e for comparison, × 38 000. A F I P neg. 78-7011-2. FIG. 3. In this micrograph only a part of a c h r o m o s o m e is artifactually altered. The centromeric section (arrows) is clumped, and few individual fibers can no longer be recognized. A strand of fused and stretched chromatin is visible at the upper left corner with only one chromatin fiber still well preserved (arrows). A strand s u c h as this should be a warning that an otherwise well-appearing preparation might contain artifacts. × 12 000, A F I P neg. 78-7011-3. FIG. 4. A c h r o m o s o m e utterly stretched lengthwise. Only a few fibers retain their original shape and diameter; this is more clearly s h o w n in the marked frame depicting the enlarged fibers (Fig. 5). × 13 000. A F I P neg. 78-7011-4. FIG. 5. Enlarged portion of Fig. 4. Most of the stretched fibers cannot be individually discerned; s o m e s e e m to have fused. This fusion is quite different from that s h o w n in Figs. 1-3. S o m e fibers, probably of c h r o m o m e r i c nature, are not stretched but m e a n d e r and loop through and above the bundle of stretched fibers. × 38 000. A F I P neg. 78-7011-5. FIG. 6. Some highly c o n d e n s e d c h r o m o s o m e s are seen side by side with a severely stretched one. A wellp r e s e r v e d E-group c h r o m o s o m e can be discerned. The stretching of the c h r o m o s o m e to the right is longitudinal as well as lateral; it has practically destroyed one chromatid. The stretched fibers are partly fused. This kind of stretching reveals that a chromatid appears to consist of fibrous material only, but it obscures or obliterates the course o f individual fibers, x 13 000. A F I P neg. 78-7011-6. Fro, 7, A c h r o m o s o m e severely stretched, both longitudinally and laterally. At n u m b e r s 1 and 2 the chrom o m e r e s are situated at a point from which the centromeric fiber connections between the sister chromatids are originating. Other n m n b e r s suggest sites for corresponding c h r o m o m e r e s on sister chromatids. The figure d e m o n s t r a t e s further the essentially fibrous nature of an entire c h r o m o s o m e and the relative mobility of chrom o m e r e s with respect to each other and to longitudinal and lateral portions as well as their variable shapes. x 13 000. A F I P neg. 78-7011-7.
30
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S t r e t c h i n g is c a u s e d by m e c h a n i c a l forces acting on the chromatin fibrils anchoring nuclei and chromosomes to the protein film at the water-air interphase of the trough used to " s p r e a d " chromosomes. It is recognizable by the relatively good p r e s e r v a t i o n of individual fibers. The stretching forces act either in the direction of the long axis of the chromosome (Figs. 4 and 5) or in directions at an angle or perpendicular to this long axis (Fig. 6). In both instances a certain amount of fusion and of smudging of the fiber profiles is recognizable, especially on comparison with the diameter and shape of normal fibers. Chromosomes stretched lengthwise offer little information beyond the realization that t h e y can be s t r e t c h e d . C h r o m o s o m e s stretched sideways yield some insight into their architecture (Fig. 7); one can frequently find the chromomeres dislodged and out of register.
stance, with gradual transitions from thinner to thicker fibers. This effect seems to occur at times selectively in only a few chromosomes of a metaphase, but is then always an indication of damage elsewhere that is not readily apparent. Moisture in the critical-point-drying process produces fiber fusion without stretching (Figs. 2 and 3). Mechanical forces cause stretching with some fusion of fibers. Individual fibers become thinner and elongated and have a straight course. The outlines are indistinct. When both moisture and stretching are encountered, bundles and strands of varying thickness may result. The same changes to smeared, bundled fibers have been observed for a variety of human cell lines and for chromatin and chromosomes in a number of other mammals, for amphibians, insects, and protozoa, and are therefore considered of general interest.
DISCUSSION
This work was completed while Dr. Bahr was an awardee of the Alexander von Humboldt Foundation, Bad Godesberg, West Germany.
C h r o m o s o m e s and implicitly nuclear chromatin are very sensitive to the manner of preparation. The unfixed but alcohol- or acetone-dehydrated samples for electron microscopy sometimes show general damage through the effects of either moisture or mechanical forces or both. The salient feature of damage is loss of individuality and therefore loss of the three-dimensionality of fibers through their fusion with others. Fused strands of chromatin give the impression of a viscous or elastic sub-
REFERENCES 1. BAHR, G. F. (1977) in YuNIs, J. J. (Ed.), Molecular Structure of Human Chromosomes, pp. 144203, Academic Press, New York. 2. BAHR, G. F., AND GOLOMB, H. M. (1974) Chrom o s o m a 46, 247-254. 3. DAVIES, H. G. (1968) J. Cell Sci. 3, 129-150. 4. Du PP,AW, E. J. (1970) DNA Chromosomes, Holt, Rinehart & Winston, Inc., New York. 5. HEUMAN, H. G. (1974) C h r o m o s o m a 47, 133-146. 6. RIS, H. (1956) J. Biophys. Biochem. Cytol. 2, 385392.
Artifacts Observed in Critical-Point-Dried Preparations of Human Chromosomes by Electron Microscopy 1 G. F. B A H R A N D W. F. E N G L E R Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306 Received May 21, 1979, and in revised form June 25, 1980 Preparations of eucaryotic c h r o m o s o m e s tend to suffer from the effects of moisture in criticalpoint drying and from mechanical stretching during preparation, resulting in fusion of fibers so that the latter are ultimately transformed into thick strands. Mechanical stretching alone is accompanied by s o m e fusion, but it m a y render insight into the architecture of c h r o m o s o m e s .
200 A in average diameter (1--6). These fibers are v e r y easily stretched and fuse readily with their neighbors; furthermore, they can be damaged during preparation by harsh fixation, dehydration, extractir/g milieu, or mechanical forces. In this article we will describe some damaged chromatin fi~ bers, as we have observed them.
The analysis of the architecture of chromosomes is currently pursued in many laboratories. Some use, as we do, the method of critical-point drying. A relatively new method of preservation or fixation applied to a biological object about which relatively little is known, such as the fine structure of the mammalian c h r o m o s o m e , might present crucial problems as to the " r e a l i t y " of the o b s e r v e d object. In the case of the mammalian chromosome, a definitive answer as to reality is made more difficult because the structural details (e.g., chromosomal constrictions and banding features) are either clearly within the limit of resolution of light microscopy or they are clearly b e y o n d its resolution, as fibers with a diameter of 200 A would be. The observation of fibers with such a dia m e t e r as the principal feature of their structure, therefore, is obviously in need of repeated confirmation. Also needed are diverse preparatory approaches and multiple observations using the same preparatory method to attest to the invariability of the fiber appearance. There is now general consensus that the fiber of chromatin in the nucleus and in chromosomes of humans is a knotty, contorted construction of about
M A T E R I A L S AND M E T H O D S H u m a n cells from tissue culture were h a r v e s t e d after a 2- to 4-hr exposure to colcemid for mitotic arrest. They were then e x p o s e d to h y p o t o n i c H a n k s ' balanced salt solution, 1:1 dilution, or to distilled water, both of which induced the cells to swell. U p o n centrifugation, s o m e of the swollen cells were placed onto the surface of water in a L a n g m u i r - t y p e trough. T h e y were picked up on carbon-coated F o r m v a r grids and critical-point-dried f r o m e t h a n o l or a c e t o n e . U n stained c h r o m a t i n was viewed in the electron microscope at 100-kV acceleration of voltage. RESULTS
The distinguishing features of criticalpoint-dried chromatin fibers considered as well preserved are (a) a distinct, sharp cont o u r in t w o - d i m e n s i o n a l images, (b) a rounded, although irregular, shape when viewed in stereopairs, (c) a relatively constant diameter at various sites in the nucleus or chromosome, and (d) a relatively uniform density (electron scattering property) along the length of the fiber. When two or more fibers cross, the electron scattering power at the crossover point is doubled or multiplied in a predictable, measurable
The opinions or assertions contained herein are the private views of the a u t h o r s and are not to be c o n s t r u e d as official or as reflecting the views of the D e p a r t m e n t o f the A r m y or the D e p a r t m e n t o f Defense. 27
0022-5320/80/100027-07502.00/0 Copyright O 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
28
BAHR AND ENGLER
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r
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o,
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~
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FIG. 1. A limited area out of a m e t a p h a s e illustrating s o m e recognizable but not well-preserved chromosomes. In the lower left corner can be seen a totally clumped c h r o m o s o m e , w h o s e fibers merge with the rest of the c h r o m o s o m a l material in the middle o f this field. N u m e r o u s particles of approximately spherical shape m a r k the e x t e n s i o n of loops and naked D N A (not visualized without staining or s h a d o w casting) into the background w h e r e they adhere to the carrier protein film essential to this type of preparation. The preservation of the c h r o m a t i n is generally severely affected. × 62 000. Inset: View of well-preserved nuclear chromatin fibers, × 100 000, A F I P neg. 78-7011-1.
A R T I F A C T S IN C H R O M O S O M E S
manner. Similarly, the transradiated mass increases when the fiber is folded back on itself or runs a course parallel to the direction of observation (on a positive print the contrast of the fiber will be seen to be increased). Features a, c, and d are illustrated in insets to Fig. l (nucleus) and Fig. 2 (chromosome). It is our experience from working with critical-point-dried c h r o m o s o m e s for the last 10 years that the normal appearance of the chromatin fiber, e.g., its diameter, is narrowly distributed (insets to Figs. 1 and 2). Foremost among the alterations fibers undergo are stretching and fusion. In the worst of instances both occur at the same time, and a c h r o m o s o m e may be so clumped that its identity as a chromosome is actually in question. Clumped and coalesced fibers are readily recognized in Figs. 1 and 2. Some affected chromosomes are better preserved than others, although riot opti-
29
really, but there can be no question as to whether they are chromosomes. It is more difficult to r e c o g n i z e the e x t e n d e d , smudged, centromeric area in the chromosome shown in Fig. 3 as an artifact, because the rest of this c h r o m o s o m e is relatively well preserved. The pattern of more or less well-preserved fiber loops emerging at the sides of chromatids documents the relatively high level o f p r e s e r v a t i o n . T h e r e is n e v e r t h e l e s s a s t r a n d o f c l u m p e d and stretched chromatin in the upper left corner of Fig. 3, signaling that this preparation contains artifacts. Only one or two fibers (arrows) retain their natural shape and proper diameter of 200 ]~. Fusion or coalescence of chromatin fibers is, as far as we can determine, chiefly due to faulty critical-point drying of the unfixed chromatin. Either incomplete removal of water from or an excess of moisture in the dried preparation causes the obliteration of individual fibers.
FIG. 2. Partial m e t a p h a s e plate. T h e c h r o m o s o m e s hang together. T h e fibrous nature is d e a r l y recognizable in m o s t of them. In the middle and lower right, c h r o m o s o m a l structure is severely affected by fiber clumping and fusion. T h e crispness of the individual fiber is lost; only a few fibers above the carrier film retain their original d i m e n s i o n s and individuality (arrows). × 10 800. Inset: View of well-preserved telomeres of a chrom o s o m e for comparison, × 38 000. A F I P neg. 78-7011-2. FIG. 3. In this micrograph only a part of a c h r o m o s o m e is artifactually altered. The centromeric section (arrows) is clumped, and few individual fibers can no longer be recognized. A strand of fused and stretched chromatin is visible at the upper left corner with only one chromatin fiber still well preserved (arrows). A strand s u c h as this should be a warning that an otherwise well-appearing preparation might contain artifacts. × 12 000, A F I P neg. 78-7011-3. FIG. 4. A c h r o m o s o m e utterly stretched lengthwise. Only a few fibers retain their original shape and diameter; this is more clearly s h o w n in the marked frame depicting the enlarged fibers (Fig. 5). × 13 000. A F I P neg. 78-7011-4. FIG. 5. Enlarged portion of Fig. 4. Most of the stretched fibers cannot be individually discerned; s o m e s e e m to have fused. This fusion is quite different from that s h o w n in Figs. 1-3. S o m e fibers, probably of c h r o m o m e r i c nature, are not stretched but m e a n d e r and loop through and above the bundle of stretched fibers. × 38 000. A F I P neg. 78-7011-5. FIG. 6. Some highly c o n d e n s e d c h r o m o s o m e s are seen side by side with a severely stretched one. A wellp r e s e r v e d E-group c h r o m o s o m e can be discerned. The stretching of the c h r o m o s o m e to the right is longitudinal as well as lateral; it has practically destroyed one chromatid. The stretched fibers are partly fused. This kind of stretching reveals that a chromatid appears to consist of fibrous material only, but it obscures or obliterates the course o f individual fibers, x 13 000. A F I P neg. 78-7011-6. Fro, 7, A c h r o m o s o m e severely stretched, both longitudinally and laterally. At n u m b e r s 1 and 2 the chrom o m e r e s are situated at a point from which the centromeric fiber connections between the sister chromatids are originating. Other n m n b e r s suggest sites for corresponding c h r o m o m e r e s on sister chromatids. The figure d e m o n s t r a t e s further the essentially fibrous nature of an entire c h r o m o s o m e and the relative mobility of chrom o m e r e s with respect to each other and to longitudinal and lateral portions as well as their variable shapes. x 13 000. A F I P neg. 78-7011-7.
30
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ARTIFACTS IN CHROMOSOMES
33
S t r e t c h i n g is c a u s e d by m e c h a n i c a l forces acting on the chromatin fibrils anchoring nuclei and chromosomes to the protein film at the water-air interphase of the trough used to " s p r e a d " chromosomes. It is recognizable by the relatively good p r e s e r v a t i o n of individual fibers. The stretching forces act either in the direction of the long axis of the chromosome (Figs. 4 and 5) or in directions at an angle or perpendicular to this long axis (Fig. 6). In both instances a certain amount of fusion and of smudging of the fiber profiles is recognizable, especially on comparison with the diameter and shape of normal fibers. Chromosomes stretched lengthwise offer little information beyond the realization that t h e y can be s t r e t c h e d . C h r o m o s o m e s stretched sideways yield some insight into their architecture (Fig. 7); one can frequently find the chromomeres dislodged and out of register.
stance, with gradual transitions from thinner to thicker fibers. This effect seems to occur at times selectively in only a few chromosomes of a metaphase, but is then always an indication of damage elsewhere that is not readily apparent. Moisture in the critical-point-drying process produces fiber fusion without stretching (Figs. 2 and 3). Mechanical forces cause stretching with some fusion of fibers. Individual fibers become thinner and elongated and have a straight course. The outlines are indistinct. When both moisture and stretching are encountered, bundles and strands of varying thickness may result. The same changes to smeared, bundled fibers have been observed for a variety of human cell lines and for chromatin and chromosomes in a number of other mammals, for amphibians, insects, and protozoa, and are therefore considered of general interest.
DISCUSSION
This work was completed while Dr. Bahr was an awardee of the Alexander von Humboldt Foundation, Bad Godesberg, West Germany.
C h r o m o s o m e s and implicitly nuclear chromatin are very sensitive to the manner of preparation. The unfixed but alcohol- or acetone-dehydrated samples for electron microscopy sometimes show general damage through the effects of either moisture or mechanical forces or both. The salient feature of damage is loss of individuality and therefore loss of the three-dimensionality of fibers through their fusion with others. Fused strands of chromatin give the impression of a viscous or elastic sub-
REFERENCES 1. BAHR, G. F. (1977) in YuNIs, J. J. (Ed.), Molecular Structure of Human Chromosomes, pp. 144203, Academic Press, New York. 2. BAHR, G. F., AND GOLOMB, H. M. (1974) Chrom o s o m a 46, 247-254. 3. DAVIES, H. G. (1968) J. Cell Sci. 3, 129-150. 4. Du PP,AW, E. J. (1970) DNA Chromosomes, Holt, Rinehart & Winston, Inc., New York. 5. HEUMAN, H. G. (1974) C h r o m o s o m a 47, 133-146. 6. RIS, H. (1956) J. Biophys. Biochem. Cytol. 2, 385392.