Journal of Microscopy, Vol. 203, Pt 1, January 2001, pp. 1–5. Received 24 October 2000; accepted 12 February 2000 SHORT COMMUNICATION GTG banding pattern on human metaphase chromosomes revealed by high resolution atomic-force microscopy S. THALHAMMER*, U. KOEHLER², R. W. STARK* & W. M. HECKL* *University Mu ¨nchen, Institute for Crystallography, Theresienstr. 41, 80333 Mu ¨nchen, Germany ²MGZ Medizinisch Genetisches Zentrum Mu ¨nchen, Bayerstr. 53, 80335 Mu ¨nchen, Germany Key words. Atomic force microscopy, GTG banding, karyotype. Summary Surface topography of human metaphase chromosomes following GTG banding was examined using high resolution atomic force microscopy (AFM). Although using a com- pletely different imaging mechanism, which is based on the mechanical interaction of a probe tip with the chromosome, the observed banding pattern is comparable to results from light microscopy and a karyotype of the AFM imaged metaphase spread can be generated. The AFM imaging process was performed on a normal 2n  46, XX karyotype and on a 2n  46, XY, t(2;15)(q23;q15) karyotype as an example of a translocation of chromosomal bands. Introduction Chromosome banding techniques have facilitated the precise identification of individual chromosomes. The GTG banding obtained by digesting the chromosomes with proteolytic trypsin followed by Giemsa staining is the most widely used in routine chromosome analysis. The inter- pretation of the GTG bands is still in progress. A direct role of Giemsa stain in producing the GTG bands was suggested (McKay, 1973). Several authors implied that chromosomes contain a pre-existing structure and this is subject of enhancement after GTG banding. But it is still unclear how this enhancement occurs (Comings, 1978; Ambros & Sumner, 1987). It is hypothesized that the differences between positive and negative GTG bands may be induced by the spatial organization of chromosomal protein and DNA. Since the invention of atomic force microscopy (AFM) (Binnig et al., 1986), high resolution imaging has been performed on various biological applications including chromosomes (Putman et al., 1992). Musio and colleagues worked on imaging the longitudinal patterns in untreated human chromosomes and imaged the chromosome structure after GTG banding (Musio et al., 1997). The detection of numerical chromosomal abnormalities of untreated chromo- somes with AFM was done by Ergu ¨n et al. (1999). In our experiments we performed AFM on human metaphase chromosomes after GTG-banding on a normal 2n  46, XX karyotype and a 2n  46,XY, t(2;15) (q23;q15) karyotype. We present data that show that reliable GTG karyotypes can be generated from AFM data. Material and methods Chromosome preparation and GTG banding Heparinized human whole blood (0.4 mL) was cultured at 37 8C for 72 h in 10 mL Gibco Chromosome Medium 1A. Cells were arrested with colchicine (10 mg mL 21 ) for 30 min. Chromosome preparations were made by incub- ating the cell suspension in 0.075 m KCl at 37 8C for 13 min, followed by a fixation step in a freshly prepared mixture of 3 : 1 methanol : acetic acid at 2 20 8C. GTG banding was performed by incubating the glass slides in a 0.05% trypsin solution (Difco) at 37 8C for 15 s, followed by rinsing the slides in phosphate-buffered saline buffer and staining in a 5% Giemsa stain for 8 min. The slides were rinsed with water and air dried. Atomic-force microscope An atomic-force microscope (AFM, Topometrix Explorer) with 130 mm xy-scan range and 10 mm z-scanner was used. It was mounted on top of an inverted microscope in order to select the metaphase spreads. For imaging the GTG q 2001 The Royal Microscopical Society 1 Correspondence: Professor W. M. Heckl, Institut fu ¨ r Mineralogie und Angewandte Kristallographie, Ludwig-Maximilians-Universita ¨t, Theresienstr. 41, 80333 Mu ¨nchen, Germany. Tel.: 149 89 2394 4331; fax: 0049 89 2394 4331; e-mail: w.heckl@lrz.uni-muenchen.de Journal of Microscopy 909 JMS MI6136 KAS 9/3/1 11:21 A LDEN