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© 1997 Oxford University Press 1662-1664

Footnote

Simultaneous detection of near-field topographic and fluorescence images of human chromosomes via scanning near-field optical/atomic-force microscopy (SNOAM)

Simultaneous detection of near-field topographic and fluorescence images of human chromosomes via scanning near-field optical/atomic-force microscopy (SNOAM) Shinichiro Iwabuchi, Hiroshi Muramatsu1, Norio Chiba1, Yasuhito Kinjo2, Yuji Murakami, Toshifumi Sakaguchi, Kenji Yokoyama and Eiichi Tamiya*

School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-12, Japan, 1Research Laboratory for Advanced Technology, Seiko Instruments Inc., 563 Takatsuka-shinden, Matsudo, Chiba 271, Japan and 2Department of Radiation Research, Tokyo Metropolitan Isotope Research Center, 2-11-1 Fukazawa, Setagaya, Tokyo 158, Japan

Received October 3, 1996; Revised and Accepted March 4, 1997

ABSTRACT

Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographical and optical images of human chromosomes using a sharp and bent optical fiber as a near-field optical probe. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. The SNOAM system does not need pretreatment of samples such as metal coating or chemical immobilization. Near-field topographic and fluorescence images provided useful information on native chromosome structure.

The previous method of scanning near-field optical microscopy (SNOM) has employed tunneling current or shear force to control the distance between the sample surface and probe (1 ,2 ). We have developed a new type of SNOM, SNOAM which can use a tapping and a non-contact mode for control the distance between the sample surface and probe in the air and liquid, respectively (3 -5 ). In this paper, the SNOAM system was first applied to simultaneous detection of topographic and fluorescence images of native human chromosomes which were obtained by tapping mode.

The metaphase chromosomes were derived from human B cell lymphoblastoid line RPMI1788 (6 ). The cells were grown in RPMI1640 medium with 10% of fetal calf serum (FCS) at 37oC. Metaphase chromosomes were obtained after addition of colcemid (final concentration 0.05 mg/ml) which gave synchronization of the cell cycle. Synchronized cells were harvested by centrifugation (500 g, 5 min). Chromosomes were prepared by the `Surface-Spreading Whole-Mount Technique' (7 ). To begin with, after centrifugation, collected cells were placed on the clean surface of distilled water with a clean platinum loop. Here, the cells burst due to osmotic pressure and then spread out rapidly over the water surface. The chromosomes were transferred to a glass coverslip by contact with the surface of the water.


Figure 1. Dynamic mode AFM (DFM) image displaying the topography of human metaphase chromosomes on a glass coverslip in air. The image area is 10 * 10 [mu]m. The dark-brown (umber) to white scale of the image is 80 nm. The height information in cyclic contact mode is calibrated just as for the height information in the conventional contact mode; by the voltages applied by the feedback amplifier to a piezoelectric scanner to keep the deflection signal constant.


Figure 2. Near-field fluorescence image of the human metaphase chromosome corresponding to (b) in Figure 1 obtained with Ar laser beam excitation ([lambda] = 488 nm: emission >520 nm). The image area is 7 * 2 [mu]m. Maximum intensity of fluorescence from chromosome was 7-fold higher than background level (0.5 * 103 mV).

All operations were done in air using the SNOAM system based on a commercially available AFM unit (model SPI 3700, Seiko Instruments Inc., Japan) which contained a non-contact AFM function and accepts a user's input signal. The system consisted of the AFM unit, an optical fiber probe, an optical source, a modulator, a detector and a lock-in amplifier. Figure 1 shows a typical topographic image of human metaphase chromosomes which are recognized as three different types of chromosomes. Generally, karyotype analysis is based on size and location of centromere and telomere regions. From a morphological view of chromosome (b) in Figure 1 , its length is ~8-10 [mu]m, and a centromere is observed on one side of the chromosome. The candidate of chromosome (b) in Figure 1 is #4 or #5. Similarly, chromosomes (a) and (c) in Figure 1 are also judged to be members of chromosome groups #6-12 and #13-15, respectively. Moers et al. reported shear force images of human chromosomes (8 ). Its spatial resolution is, however, defined not only by the radius of the tip of the probe but also by the amplitude of variation. Therefore the resolution of the SNOAM image is a better method than that of a shear-force feedback microscope (4 ). SYBRTMGreen I (excitation at 497 nm, emission at 520 nm) was used as a fluorescent intercalater combined with DNA strands. The fluorescence images were detected at 520 nm emission with 488 nm Argon laser beam excitation. Figure 2 shows the round shape of the fluorescent chromosome corresponding to (b) in Figure 1 . Topographic images clearly indicated duplicated structure on the metaphase chromosome, while fluorescence images were a different shape probably because it depended on the combination of SYBRTMGreen I and chromosome DNA. Signal light from chromosomes was corrected by an objective lens and separated by a dichroic mirror to the CCD camera and detectors. A photomultiplier and intensified CCD camera with spectrometer were connected as detectors. Fluorescence spectra obtained from the microscopic area were the same as that of SYBRTMGreen I intercalated with the DNA strand. Structural analysis of specific region of chromosomes will be possible using the FISH technique (9 ). Atomic force images have some artefacts (Fig. 1 ), however they can be corrected by comparison with the fluorescence image. The resolution of conventional far-field optical microscopy is theoretically over half of the wavelength. Although electron microscopy provides better resolution it cannot allow observation without preparative procedures, such as fixation, staining and vacuum evaporation. In this regard, SNOAM will be a useful application in biology.

ACKNOWLEDGEMENTS

We are grateful to Mr Mark Peterson for improvement of the English in this manuscript. This study was supported in part by the Original Industrial Technology R&D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

REFERENCES

1 Dürig,U.T., Pohl,D.W. and Rohner,F. (1986) Appl. Phys. Lett., 59, 3318-3327.

2 Betzig,E. and Chichester,R.J. (1993) Science, 262, 1422-1425.

3 Muramatsu,H., Chiba,N., Ataka,T., Monobe,H. and Fujihira,M. (1995) Ultramicroscopy, 57, 141-146.

4 Fujihira,M., Monobe,H., Muramatsu,H. and Ataka,T. (1995) Ultramicroscopy, 57, 118-123.

5 Muramatsu,H., Chiba,N., Homma,K., Nakajima,K., Ataka,T., Ohta,S., Kusumi,A. and Fujihira,M. (1995) Appl. Phys. Lett., 66, 3245-3247.

6 Huang,C.C. and Moore,G.E. (1969) J. Natl. Cancer. Inst., 43, 1119-1128.

7 Watanabe,M. and Tanaka,N. (1972) Jpn. J. Genetics, 47, 1-18.

8 Moers,M.H.P., Kalle,W.H.J., Ruiter,A.G.T., Wiegant,J.C.A.G., Raap,A.K., Greve,J., de Grooth,B.G. and van Hulst,N.F. (1996) J. Microscopy, 182, 40-45.

9 Lichter,P. and Ward,D.C. (1990) Nature, 345, 93-94. MEDLINE Abstract

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*To whom correspondence should be addressed. Tel: +81 761 51 1660; Fax: +81 761 51 1665; Email: tamiya@jaist.ac.jp
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