Atomic Force Microscope , a new tool for cytogenetic studies

The atomic force microscopy (AFM) has been considered, in recent years, an instrument of high-resolution study of the different cytogenetic troubles and it was found a potential diagnostic tool designed to overcome the resolution limits associated with the light microscopy in the cytogenetic diagnosis and research. The AFM is equipped with a sharp probe tip that scans across a solid sample by monitoring the forces of interaction between the tip and the surface of the sample. This creates three-dimensional information about the sample topography with an horizontal resolution and a vertical resolution of nanometer-scale. These features allow to investigate different chromosome aberrations in liquid and air environments as well as in vacuum. The chromosomal morphologies showing a thickness modulation and these thicker regions could be associated with GTG bands (G-banding by trypsin with Giemsa). The banding pattern on each chromosome is exclusive, allowing each chromosome to be numbered and identified (species’s karyotype). Banding shows that chromosomes are organized into a pattern of regions, differing each in gene density, time of replication, and base composition. The Giemsa dark bands (facultative heterochromatic bands, AT rich) replicate later than the R-bands (reverse of G-bands, euchromatic and GC rich) where most of the mapped human genes are located. Therefore, AFM could integrate the information obtained by banding techniques with nanometer-resolved topographic data images of chromatin structures. The use of AFM furthermore overcomes the resolving limits of traditional microscopy in detecting chromosomal aberrations, such as small deletions, translocations and duplications without any staining techniques and therefore generating fewer artefacts. The chromosomes also differ in the volume extent. The relationship of the bands with the topography and the determination of the volumes may be a new diagnostic tool. The volume of chromosomes may be related, indeed, to the amount of DNA and the amplifications or losses of genetic material can be accurately determined. The AFM can be used also to easy manipulate chromosomes at high resolution; chromosomal nanodissection provides a direct approach for isolating DNA from any cytogenetically recognizable region at the submicron scale under highresolution image control. The combination of the nanomechanics tool box and biochemical techniques like Polymerase Chain Reaction (PCR) shows enormous potential for the future development of single molecule techniques, ranging from applications in DNA mechanics to cytogenetic studies and biochip development. The nanodissected material can be used for a variety of applications in the field of genomic research as well as establishing probes for fluorescence in situ hybridization (FISH), genetic linkage map and physical map construction. The availability of increasingly accurate probes requires a higher resolution for the precise detection of fluorescence signals. However, high-resolution analysis of fluorescent signals is notionally limited by the 300-nm resolution optical limit of light microscopy. The Scanning Nearfield Optical Microscope (SNOM), a microscopic technique that breaks the far field resolution limit by exploiting the properties of evanescent waves, and AFM can simultaneously obtain fluorescent and topographic images with nanometerscale resolution in one scan and offer useful information about the chromosome structure and fluorescent intensity.