Journal of Microscopy, Vol. 168, P t 3, December 1992, p p . 239-247. Received 30June 1992; accepted 3 August 1992
High-resolution imaging of chromosome-related structures by atomic force microscopy
by B A R TG. D E G R O O Tand H C O N S T A NA. T J. P U T M A NBiophysical , Technique, Department of Applied Physics, University of Twente, PO Box 217, 7500 A E Enschede, The Netherlands
K E Y W O R D S . AFM, SFM, chromosomes, G-banding, in situ hybridization, synaptonemal complex, optical microscope.
SUMMARY
An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. 1ma:-es of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein. INTRODUCTION
The invention of the scanning tunnelling microscope (STM) in 1981 by 3innig et al. (1982) brought about a revolution in surface science because single atoms could be resolved. Four years later, Binnig, Quate and Gerber invented the atomic force microscope (AFM) (Binnig et al., 1986). With the AFM atomic resolution can also be achieved, but unlike the STM, the AFM can be applied directly to non-conducting surfaces. Furthermore, the AFM can be operated under ambient conditions: no ultrahigh vacuum is needed. This, together with the fact that the AFM can give images of objects in an aqueous environment (Drake et al., 1989), opens up exciting possibilities for biological applications. With the AFM the surface of the sample is scanned with a very sharp tip mounted on a small cantilever with a low force constant. The force between tip and sample causes a displacement of the cantilever which is measured with high accuracy. With a feedback loop the force is kept constant by changing the height of the sample during a raster scan. This height signal is displayed as a function of sample position, and yields an image of the surface of the object. The AFM has been applied to a variety of biological cells and subcellular objects
0 1992 The Royal Microscopical Society 239
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including erythrocytes (Gould et al., 1990), lymphocytes (Butt et al., 1991), gap junctions (Hoh et al., 1991) and chromosomes (De Grooth et al., 1992; Putman et al., 1992~).Images of enzymes (Edstrom et al., 1990) and DNA (Bustamante et al., 1992; Hansma et al., 1992)have been successfully obtained. For a review of applications of the AFM to biological samples see Hoh & Hansma (1992). For a typical AFM image, a scan takes about 30 s with a maximum scan area of about 20 x 20 pm. Although this seems reasonably fast, if it is not certain that the object of interest is located inside this area, the total time needed to obtain an image of the object of interest can be quite long. T o overcome this problem an AFM has been developed that is integrated with a conventional optical microscope. This greatly reduces the time needed to locate the object and ‘in addition’ it provides the opportunity to make a direct comparison between the optical and AFM images. This article reviews some of the results that have been obtained with this instrument in the field of structural chromosome research. Part of this research is published elsewhere (De Grooth et al., 1992; Putman et al., 1992~). MATERIALS A N D METHODS
Sample preparation Metaphase chromosomes were isolated from Chinese hamster lung cells (V78) using the standard procedure for optical microscopy (Lee & Bahr, 1983). Exponentially growing cells were arrested in the metaphase by colcemid. After hypotonic treatment the cells were fixed in methanol and acetic acid. G-banding was obtained by treating the metaphase chromosomes with trypsin followed by staining with Giemsa (Seabright, 1971). Metaphase chromosomes were produced from phytomaglutinin-stimulated normal human peripheral blood lymphocyte cultures to enable the visualization of in situ hybridization probes. In situ hybridization with biotinated pUCl.77 DNA was conducted according to Wiegant et al. (1991). It was detected with one layer of peroxidase conjugated to avidin followed by the diaminebenzidine (DAB) reaction (Graham & Karnovsky, 1966). Synaptonemal complexes were isolated from rat spermatocytes according to the method described by Heyting et al. (1985). The atomic force microscope with integrated optical microscope A schematic view of the AFM combined with an optical microscope is shown in Fig. 1 (Putman et al., 1992~). The sample is placed on a standard microscope glass, which is mounted on the hollow piezo-tube (Staveley Sensors Inc., Hartford, Conn., U.S.A.) using a small vacuum chamber that facilitates the easy and quick exchange of samples (Putman et al., 1992d). By applying suitable voltages across the four segments of this tube, the sample can be moved in three dimensions (Binnig & Smith, 1986). Inside the piezo-tube a small lens, normally used in a compact disc player, is mounted on an xystage. The lens has a numerical aperture of 0.4 and can be moved up and down in the tube for focal adjustment. The lens serves as the objective for the optical epiillumination microscope. The upper part of the instrument contains the AFM unit. The microfabricated cantilevers with integrated pyramidal tips (Albrecht et al., 1990)were purchased from Park Scientific Instruments (Sunnyvale, Calif., U.S.A.). A change in force between the tip and the sample causes a displacement of the cantilever which is measured using the optical beam deflection method (Meyer & Amer, 1988; Putman et al., 1992a). During the image acquisition, deviations from a pre-set cantilever displacement are compensated by adjusting the z-position of the sample using a feedback circuit. Due to this feedback system the force exerted by the tip on the sample remains at an average low
AFM of chromosomes
241
m\\ // Tip
h
Moni'ur-'
m
-
1
I
t @
M
CCD-camera
3
Monimr-2
Data acquisition
u
Fig. 1. Schematic of the combined atomic force and optical microscope. With the inverted optical microscope (consisting of Hg-lamp for illumination, beamsplitter (BS), CD-lens, mirror (M), CCD-camera and Monitor-1) the sample and the tip can be observed simultaneously. The AFM tip is in line with the CD-lens which can be translated with XY-2. T h e sample on top of the piezo-tube can be moved using XY-1. The displacement of the tip is detected by measuring the change in deflection of the laser beam with a positionsensitive detector (PSD). Images are acquired with an HP9000-based system and are shown on Monitor-2. For a detailed description see Putman er al. (1992~).
level of typically N in air and N in liquid (Weisenhorn et al., 1989), even for relatively high objects such as cells. The feedback signal is essentially proportional to the local height of the sample. However, because of the limited bandwidth of the feedback loop, only slowly varying changes in the topography of the surface are perfectly compensated. The error signal, due to the remaining movement of the cantilever, gives the high (spatial) frequency information of the sample in the scan direction. Both signals can be used for displaying the image (Putman e t al., 1992b). The images shown in this article use the high-frequency information, the so-called error signal mode. For the high-resolution AFM image of the chromosome structure shown in Fig. 4, a fine needle of carbon was grown on top of the normal pyramidal tip using the scanning electron microscope (SEM) (Keller & Chih-Chung, 1992). An SEM image of the resulting tip is shown in Fig. 2. By an initial adjustment (using the XY-2 stage) the tip is positioned in the middle of the optical image. A sample on the microscope glass can now be observed with the optical microscope and moved with the mechanical translation stage XY-1. After the object of interest is found, the AFM tip is brought into contact with the sample and a high-resolution AFM image can be obtained. The acquisition time of an AFM image (5 12 x 5 12 pixels) is between 15 s and 2 min. RESULTS A N D D I S C U S S I O N
A typical AFM image of a metaphase chromosome isolated from Chinese hamster chromosomes and prepared according to standard procedures for optical microscopy is shown in Fig. 3(a). The height of the chromatids of air-dried samples is only about 50 nm. The structures directly surrounding the chromosome are always observed. The height of these structures is about 10 nm and they are thought to originate from the
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B . G . De Grooth and C . A . J . Putman
Fig. 2. SEMimage of a detail ofthe Si3N4 cantilever with tip. On top of the original pyramidal tip a fine needle with a radius of less than 10 nm at the apex was grown by carbon deposition using SEM (Keller & ChihChung, 1992). Scan range: 12 x 8 pm.
chromosomes. Most chromosomes display a more or less pronounced groove pattern on their arms, in agreement with earlier optical and electron microscopical observations (Allen et al., 1988).The height of the chromosomes increased dramatically to about 300 nm when immersed in liquid. This is illustrated in Fig. 3(b), where the same chromosome displayed in Fig. 3(a) was measured in phosphate-buffered saline (PBS). Images obtained with a smaller scan range using the sharpened tip (see Fig. 2) revealed details of about 30 nm in size (right-hand side of Fig. 4). Although it is tempting to ascribe these structures to the 30-nm chromatin fibre, the preparation method used should be improved (e.g. by using critical-point drying) before drawing any definitive conclusions. Figure 5 shows that the AFM could be used to study the processes involved in chromosome banding. The well known, but not completely understood, G-bands can be observed with an optical microscope when trypsin-treated chromosomes are stained with Giemsa. The AFM image of these chromosomes displays variations in height that correlate well with the optical bands. This is illustrated in the inset to Fig. 5, where the optical and the AFM images of the same chromosome are compared. The observations confirm earlier SEM measurements (Allen et al., 1988). With the AFM, however, the height of the material remaining can be measured directly. Depending on the duration of the trypsin treatment this height can be surprisingly low. After full treatment the highest features have a height of about 50 nm whereas in most places the chromosome cannot be distinguished from its surroundings. We have recently succeeded in imaging the location of an in situ hybridization probe on a human chromosome by AFM ( C . A. J. Putman et al., unpublished observation). The chromosomes were hybridized with the biotinated pUCl.77 DNA probe. Incubation with avidin-conjugated peroxidase and DAB resulted in the deposition of material in the direct vicinity of the probe. The AFM image indicates that the location
AFM of chromosomes
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Fig. 3. AFM images of a metaphase chromosome isolated from Chinese hamster lung cells. Imaged in (a) air and (b) PBS. Scan range: 12 x 8 pm.
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Fig. 4. Detailed scan of a Chinese hamster metaphase chromosome using the super tip shown in Fig. 2. Scan range: 3 x 2 pm.
Fig. 5. G-banded Chinese hamster chromosome obtained by trypsin treatment and Giemsa staining. Inset: comparison of the AFM image (left, contrast reversed) and normal optical image (right) of the same chromosome. Scan range: 9 x 6 pm.
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Fig. 6. AFM image of a human chromosome after in situ hybridizationwith biotinated pUCl.77. The probe was visualized by incubation with peroxidase conjugated to avidin, followed by the diaminebenzidine reaction. The bumps near the centromer are interpreted as being caused by the deposition of enzymatic reaction product near the probe. Scan range: 7.5 x 5 pm.
of the DNA probe can be determined with an accuracy of about 100 nm (Fig. 6). Thus the AFM could be helpful in the area of gene mapping on chromosomes. Our final example is an image of the synaptonemal complex isolated from rat spermatocytes (Fig. 7). These structures are formed between homologous chromosomes during meiotic prophase and mediate the chromosome pairing, genetic recombination and disjunction (Gillies, 1975). The image in Fig. 7 clearly shows the two slightly twisted lateral elements and in some places the central element. The terminal dot at the upper right-hand corner of the image corresponds to the point of attachment to the nuclear envelope. CONCLUSIONS
Of the recently developed scanning probe microscopy techniques the AFM proves at this moment to be the most useful method for biological applications. In this article we have presented a brief overview of some of our recent results on chromosome-related structures. For these objects, the incorporation of an optical microscope was almost indispensable. The technique enables images of a variety of objects to be obtained quickly with a resolution that, under routine conditions, is about one order of magnitude better than with the optical microscope. Obvious disadvantages of the technique are that only surface topography can be measured and that the object has to be firmly attached to the surface. The possibility of acquiring high-resolution images in a liquid environment is a unique feature of the AFM which we are now just starting to survey.
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Fig. 7. AFM image of the synaptonemal complex isolated from rat. Scan range: 7.5x 5 p n ACKNOWLEDGMENTS
We thank Niek van Hulst, Kees van der Werf, Geeske van Oort, Jan Greve, Ton Raap and Joop Wiegant (in situ hybridization), and Axel Dietrich and Jan van Marle (synaptonemal complex) for valuable contributions, and Bert Otter for skilful SEM work. This work was supported by the Netherlands Organization for Scientific Research, NWO. REFERENCES Albrecht, T.R., Alkamine, S., Carver, T.E. & Quate, C.F. (1990)Microfabrication of cantilever styli for the atomic force microscope. 3. Vac. Sci. Technol. A8, 3386-3396. Allen, T.D., Jack, E.M. & Harrison, C.J. (1988)The three-dimensional structure of human metaphase chromosomes determined by scanning electron microscopy. Chromosomes and Chromarin (ed. by K. W. Adolph), Vol. 11, pp. 51-72. CRC Press, Fla., U.S.A. Binnig, G., Rohrer, H. & Gerber, Ch. (1982)Surface studies with scanning tunneling microscopy. Phys. Rev. Lett. 49, 57-61. Binnig, G. & Smith, D.P.E. (1986) Single-tube three-dimensional scanner for scanning tunneling microscopy. Rev. Sci Instrum. 57, 1688-1689. Binnig, G., Quate, C.F. & Gerber, Ch. (1986)Atomic force microscope. Phys. Rev. Lett. 56, 930-933. Butt, H.-J., Wolff, E.K., Gould, S.A.C., Dixon Northern, B., Peterson, C.M. & Hansma, P.K. (1991) Imaging cells with the atomic force microscope. 3. Struct. Biol. 105, 54-61. Bustamante, C., Vesenka, J., Tang, C.L., Rees, W., Guthold, M. & Keller, R. (1992) Circular DNA molecules imaged in air by scanning force microscopy. Biochemistry, 31, 22-26. De Grooth, B.G., Putman, C.A.J., Van der Werf, K.O., Van Hulst, N.F., Van Oort, G. & Greve, J. (1992) Chromosome structure investigated with the atomic force microscope. SPIE, 1639, 205-21 1. Drake, B., Prater, C.B., Weisenhorn, A.L., Gould, S.A.C., Albrecht, T.R., Quate, C.F., Cannell, D.S., Hansma, H.G. & Hansma, P.K. (1989)Imaging crystals, polymers and processes in water with the atomic force microscope. Science, 243, 1586-1589. Edstrom, R.D., Meinke, M.H., Yang, X., Yang, R., Elings, V. & Evans, D.F. (1990)Direct visualisation of
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phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy. Biophys. 3. 58, 1437-1448. Gillies, C.B. (1975) Synaptonemal complex and chromosome structure. Ann. Rev. Genet. 9,91-109. Gould, S.A.C., Drake, B., Prater, C.B., Weisenhorn, A.L., Manne, S., Hansma, H.G., Hansma, P.K., Massie, J., Longmire, M., Elings, V., Dixon Northern, B., Mukergee, B., Peterson, C.M., Stoeckenius, W., Albrecht, T.R. & Quate, C.F. (1990) From atoms to integrated circuit chips, blood cells and bacteria with the atomic force microscope. J. Vuc. Sci. Technol. AS, 369-372. Graham, R.C. & Karnovsky, H.J. (1966) The early stage of absorption of injected horse radish peroxidase in the proximal tubules of mouse kidney. 3. Histochem. Cytochem. 14,291-302. Hansma, H.G., Vesenka, J., Siegerist, C., Kelderman, G., Morrett, H., Sinsheimer, R.L., Elings, V., Bustamante, C. & Hansma, P.K. (1992) Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science, 256, 1180-1 184. Heyting, C., Dietrich, A.J.J., Redeker, E.J.W. & Vink, A.C.G. (1985) Structure and composition of synaptonemal complexes isolated from rat spermatocytes. Eur. J. Cell. Biol. 36, 307-314. Hoh, J.H., Lal, R., John, S.A., Revel, J.-P. & Arnsdorf, M.F. (1991) Atomic force microscopy and the dissection of gap junctions. Science, 253, 1405-1408. Hoh, J.H. & Hansma, P.K. (1992) Atomic force microscopy for high resolution imaging in cell biology. Trends Cell Biol. 2, 208-213. Keller, D. & Chih-Chung, C. (1992) Imaging steep, high structures by scanning force microscopy with electron beam deposited tips. Surf.Sci. 268, 333-339. Lee, J.C.K. & Bahr, G.F. (1983) Microfluorometric studies on chromosomes. Chromosomu, 88,374-376. Meyer, G .& h e r , N. (1988) Novel optical approach to atomic force microscopy. Appl. Phys. Lett. 53,24002402. Putman, C.A.J., De Grooth, B.G., Van Hulst, N.F. & Greve, J. (1992a) A detailed analysis of the optical beam deflection technique for use in atomic force microscopy. J. Appl. Phys. 72, 6-12. Putman, C.A.J., Van der Werf, K.O., De Grooth, B.G., Van Hulst, N.F., Greve, J. & Hansma, P.K. (1992b) New imaging mode in atomic force microscopy based on the error signal. SPIE, 1639, 198-204. Putnam, C.A.J., Van der Werf, K.O., De Grooth, B.G., VanHulst, N.F., Segerink, F.B. & Greve, J. (1992~) Atomic force microscope with integrated optical microscope for biological applications. Rev. Sci. Instrum. 63, 1914-1917. Putnam, C.A.J., Van der Werf, K.O., Van Oort, G., De Grooth, B.G., Van Hulst, N.F. & Greve, J. (1992d) Vacuum chamber for sample attachment in atomic force microscopy. Rev. Sci. Instrum. 63,1914-1915. Seabright, M. (1971) A rapid banding technique for human chromosomes. Lancet, 297,971-972. Weisenhorn, A.L., Hansma, P.K., Albrecht, T.R. & Quate, C.F. (1989) Forces in atomic force microscopy in air and water. Appl. Phys. Lett. 54, 2651-2653. Wiegant, J., Ried, Th., Van der Ploeg, M., Nederhof, P.M., Tanke, H.J. & Raap, A.K. (1991) I n situ hybridization with fluoresceinated DNA. Nucleic Acids Res. 19,3237-3241.
High-resolution imaging of chromosome-related structures by atomic force microscopy
by B A R TG. D E G R O O Tand H C O N S T A NA. T J. P U T M A NBiophysical , Technique, Department of Applied Physics, University of Twente, PO Box 217, 7500 A E Enschede, The Netherlands
K E Y W O R D S . AFM, SFM, chromosomes, G-banding, in situ hybridization, synaptonemal complex, optical microscope.
SUMMARY
An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. 1ma:-es of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein. INTRODUCTION
The invention of the scanning tunnelling microscope (STM) in 1981 by 3innig et al. (1982) brought about a revolution in surface science because single atoms could be resolved. Four years later, Binnig, Quate and Gerber invented the atomic force microscope (AFM) (Binnig et al., 1986). With the AFM atomic resolution can also be achieved, but unlike the STM, the AFM can be applied directly to non-conducting surfaces. Furthermore, the AFM can be operated under ambient conditions: no ultrahigh vacuum is needed. This, together with the fact that the AFM can give images of objects in an aqueous environment (Drake et al., 1989), opens up exciting possibilities for biological applications. With the AFM the surface of the sample is scanned with a very sharp tip mounted on a small cantilever with a low force constant. The force between tip and sample causes a displacement of the cantilever which is measured with high accuracy. With a feedback loop the force is kept constant by changing the height of the sample during a raster scan. This height signal is displayed as a function of sample position, and yields an image of the surface of the object. The AFM has been applied to a variety of biological cells and subcellular objects
0 1992 The Royal Microscopical Society 239
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including erythrocytes (Gould et al., 1990), lymphocytes (Butt et al., 1991), gap junctions (Hoh et al., 1991) and chromosomes (De Grooth et al., 1992; Putman et al., 1992~).Images of enzymes (Edstrom et al., 1990) and DNA (Bustamante et al., 1992; Hansma et al., 1992)have been successfully obtained. For a review of applications of the AFM to biological samples see Hoh & Hansma (1992). For a typical AFM image, a scan takes about 30 s with a maximum scan area of about 20 x 20 pm. Although this seems reasonably fast, if it is not certain that the object of interest is located inside this area, the total time needed to obtain an image of the object of interest can be quite long. T o overcome this problem an AFM has been developed that is integrated with a conventional optical microscope. This greatly reduces the time needed to locate the object and ‘in addition’ it provides the opportunity to make a direct comparison between the optical and AFM images. This article reviews some of the results that have been obtained with this instrument in the field of structural chromosome research. Part of this research is published elsewhere (De Grooth et al., 1992; Putman et al., 1992~). MATERIALS A N D METHODS
Sample preparation Metaphase chromosomes were isolated from Chinese hamster lung cells (V78) using the standard procedure for optical microscopy (Lee & Bahr, 1983). Exponentially growing cells were arrested in the metaphase by colcemid. After hypotonic treatment the cells were fixed in methanol and acetic acid. G-banding was obtained by treating the metaphase chromosomes with trypsin followed by staining with Giemsa (Seabright, 1971). Metaphase chromosomes were produced from phytomaglutinin-stimulated normal human peripheral blood lymphocyte cultures to enable the visualization of in situ hybridization probes. In situ hybridization with biotinated pUCl.77 DNA was conducted according to Wiegant et al. (1991). It was detected with one layer of peroxidase conjugated to avidin followed by the diaminebenzidine (DAB) reaction (Graham & Karnovsky, 1966). Synaptonemal complexes were isolated from rat spermatocytes according to the method described by Heyting et al. (1985). The atomic force microscope with integrated optical microscope A schematic view of the AFM combined with an optical microscope is shown in Fig. 1 (Putman et al., 1992~). The sample is placed on a standard microscope glass, which is mounted on the hollow piezo-tube (Staveley Sensors Inc., Hartford, Conn., U.S.A.) using a small vacuum chamber that facilitates the easy and quick exchange of samples (Putman et al., 1992d). By applying suitable voltages across the four segments of this tube, the sample can be moved in three dimensions (Binnig & Smith, 1986). Inside the piezo-tube a small lens, normally used in a compact disc player, is mounted on an xystage. The lens has a numerical aperture of 0.4 and can be moved up and down in the tube for focal adjustment. The lens serves as the objective for the optical epiillumination microscope. The upper part of the instrument contains the AFM unit. The microfabricated cantilevers with integrated pyramidal tips (Albrecht et al., 1990)were purchased from Park Scientific Instruments (Sunnyvale, Calif., U.S.A.). A change in force between the tip and the sample causes a displacement of the cantilever which is measured using the optical beam deflection method (Meyer & Amer, 1988; Putman et al., 1992a). During the image acquisition, deviations from a pre-set cantilever displacement are compensated by adjusting the z-position of the sample using a feedback circuit. Due to this feedback system the force exerted by the tip on the sample remains at an average low
AFM of chromosomes
241
m\\ // Tip
h
Moni'ur-'
m
-
1
I
t @
M
CCD-camera
3
Monimr-2
Data acquisition
u
Fig. 1. Schematic of the combined atomic force and optical microscope. With the inverted optical microscope (consisting of Hg-lamp for illumination, beamsplitter (BS), CD-lens, mirror (M), CCD-camera and Monitor-1) the sample and the tip can be observed simultaneously. The AFM tip is in line with the CD-lens which can be translated with XY-2. T h e sample on top of the piezo-tube can be moved using XY-1. The displacement of the tip is detected by measuring the change in deflection of the laser beam with a positionsensitive detector (PSD). Images are acquired with an HP9000-based system and are shown on Monitor-2. For a detailed description see Putman er al. (1992~).
level of typically N in air and N in liquid (Weisenhorn et al., 1989), even for relatively high objects such as cells. The feedback signal is essentially proportional to the local height of the sample. However, because of the limited bandwidth of the feedback loop, only slowly varying changes in the topography of the surface are perfectly compensated. The error signal, due to the remaining movement of the cantilever, gives the high (spatial) frequency information of the sample in the scan direction. Both signals can be used for displaying the image (Putman e t al., 1992b). The images shown in this article use the high-frequency information, the so-called error signal mode. For the high-resolution AFM image of the chromosome structure shown in Fig. 4, a fine needle of carbon was grown on top of the normal pyramidal tip using the scanning electron microscope (SEM) (Keller & Chih-Chung, 1992). An SEM image of the resulting tip is shown in Fig. 2. By an initial adjustment (using the XY-2 stage) the tip is positioned in the middle of the optical image. A sample on the microscope glass can now be observed with the optical microscope and moved with the mechanical translation stage XY-1. After the object of interest is found, the AFM tip is brought into contact with the sample and a high-resolution AFM image can be obtained. The acquisition time of an AFM image (5 12 x 5 12 pixels) is between 15 s and 2 min. RESULTS A N D D I S C U S S I O N
A typical AFM image of a metaphase chromosome isolated from Chinese hamster chromosomes and prepared according to standard procedures for optical microscopy is shown in Fig. 3(a). The height of the chromatids of air-dried samples is only about 50 nm. The structures directly surrounding the chromosome are always observed. The height of these structures is about 10 nm and they are thought to originate from the
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B . G . De Grooth and C . A . J . Putman
Fig. 2. SEMimage of a detail ofthe Si3N4 cantilever with tip. On top of the original pyramidal tip a fine needle with a radius of less than 10 nm at the apex was grown by carbon deposition using SEM (Keller & ChihChung, 1992). Scan range: 12 x 8 pm.
chromosomes. Most chromosomes display a more or less pronounced groove pattern on their arms, in agreement with earlier optical and electron microscopical observations (Allen et al., 1988).The height of the chromosomes increased dramatically to about 300 nm when immersed in liquid. This is illustrated in Fig. 3(b), where the same chromosome displayed in Fig. 3(a) was measured in phosphate-buffered saline (PBS). Images obtained with a smaller scan range using the sharpened tip (see Fig. 2) revealed details of about 30 nm in size (right-hand side of Fig. 4). Although it is tempting to ascribe these structures to the 30-nm chromatin fibre, the preparation method used should be improved (e.g. by using critical-point drying) before drawing any definitive conclusions. Figure 5 shows that the AFM could be used to study the processes involved in chromosome banding. The well known, but not completely understood, G-bands can be observed with an optical microscope when trypsin-treated chromosomes are stained with Giemsa. The AFM image of these chromosomes displays variations in height that correlate well with the optical bands. This is illustrated in the inset to Fig. 5, where the optical and the AFM images of the same chromosome are compared. The observations confirm earlier SEM measurements (Allen et al., 1988). With the AFM, however, the height of the material remaining can be measured directly. Depending on the duration of the trypsin treatment this height can be surprisingly low. After full treatment the highest features have a height of about 50 nm whereas in most places the chromosome cannot be distinguished from its surroundings. We have recently succeeded in imaging the location of an in situ hybridization probe on a human chromosome by AFM ( C . A. J. Putman et al., unpublished observation). The chromosomes were hybridized with the biotinated pUCl.77 DNA probe. Incubation with avidin-conjugated peroxidase and DAB resulted in the deposition of material in the direct vicinity of the probe. The AFM image indicates that the location
AFM of chromosomes
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Fig. 3. AFM images of a metaphase chromosome isolated from Chinese hamster lung cells. Imaged in (a) air and (b) PBS. Scan range: 12 x 8 pm.
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Fig. 4. Detailed scan of a Chinese hamster metaphase chromosome using the super tip shown in Fig. 2. Scan range: 3 x 2 pm.
Fig. 5. G-banded Chinese hamster chromosome obtained by trypsin treatment and Giemsa staining. Inset: comparison of the AFM image (left, contrast reversed) and normal optical image (right) of the same chromosome. Scan range: 9 x 6 pm.
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Fig. 6. AFM image of a human chromosome after in situ hybridizationwith biotinated pUCl.77. The probe was visualized by incubation with peroxidase conjugated to avidin, followed by the diaminebenzidine reaction. The bumps near the centromer are interpreted as being caused by the deposition of enzymatic reaction product near the probe. Scan range: 7.5 x 5 pm.
of the DNA probe can be determined with an accuracy of about 100 nm (Fig. 6). Thus the AFM could be helpful in the area of gene mapping on chromosomes. Our final example is an image of the synaptonemal complex isolated from rat spermatocytes (Fig. 7). These structures are formed between homologous chromosomes during meiotic prophase and mediate the chromosome pairing, genetic recombination and disjunction (Gillies, 1975). The image in Fig. 7 clearly shows the two slightly twisted lateral elements and in some places the central element. The terminal dot at the upper right-hand corner of the image corresponds to the point of attachment to the nuclear envelope. CONCLUSIONS
Of the recently developed scanning probe microscopy techniques the AFM proves at this moment to be the most useful method for biological applications. In this article we have presented a brief overview of some of our recent results on chromosome-related structures. For these objects, the incorporation of an optical microscope was almost indispensable. The technique enables images of a variety of objects to be obtained quickly with a resolution that, under routine conditions, is about one order of magnitude better than with the optical microscope. Obvious disadvantages of the technique are that only surface topography can be measured and that the object has to be firmly attached to the surface. The possibility of acquiring high-resolution images in a liquid environment is a unique feature of the AFM which we are now just starting to survey.
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B . G. De Grooth and C . A . J . Putman
Fig. 7. AFM image of the synaptonemal complex isolated from rat. Scan range: 7.5x 5 p n ACKNOWLEDGMENTS
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