Motoharu Shichiri

Nano-scale imaging of chromosomes and DNA by scanning near-field optical/atomic force microscopy

Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.

Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.

Analysis by atomic force microscopy of morphological changes in barley chromosomes during FISH treatment.

We employed atomic force microscopy (AFM) to examine structural changes in barley chromosomes during the four steps of standard FISH processes. Rehydration and dehydration with alcohol accompanying RNase treatment increased chromosome arm width and decreased chromosome height about 50%. Subsequent heat denaturation reduced chromosome height further. These three-dimensional structural changes of the chromosomes were substantial, but the FISH signal produced by the hybridization of fluorescent probes was clear when observed by a fluorescence microscope. In higher-magnification images, we observed granular structures considered to represent the chromatin fiber on the surface of the chromosomes in each FISH protocol step. These our results indicate that FISH treatments result in severe damage of the three-dimensional higher-order structures of the chromosomes, although nano-structures, such as nucleosome and chromatin fibers, remain intact and relatively unaffected.

AFM picking-up manipulation of the metaphase chromosome fragment by using the tweezers-type probe.

We have studied the development of a new procedure based on atomic force microscopy (AFM) for the analysis of metaphase chromosome. The aim of this study was to obtain detailed information about the specific locations of genes on the metaphase chromosome. In this research, we performed the manipulation of the metaphase chromosome by using novel AFM probes to obtain chromosome fragments of a smaller size than the ones obtained using the conventional methods, such as glass microneedles. We could pick up the fragment of the metaphase chromosome dissected by the knife-edged probe by using our tweezers-type probe.

The scanning probe microscope as a novel genomic analysis tool

We have developed a novel and efficient genomic analysis tool that combines scanning probe microscopy (SPM) and image processing with molecular biology techniques to accelerate genomic research. To examine the correlation between chromosome volume and DNA content, we scanned human metaphase chromosome sets with an atomic force microscope to examine the chromosome volume distribution. We found that the chromosome volume distribution agreed with DNA length distribution (obtained from a public database), and that the short arm to long arm volume ratio showed good agreement with the genomic position of the centromere. We were also able to predict the genomic position of an arbitrary gene marker with high accuracy by combining a scanning near-field optical/atomic force microscope and image processing techniques using fluorescence in situ hybridization. Thus, a novel SPM-based system developed here will be an effective tool to rapidly and accurately map DNA markers and construct physical map, which contributes to the advancement of genomic science.

Nanometer-Scale Dissection of Chromosomes by Atomic Force Microscopy Combined with Heat-Denaturing Treatment

We have developed a method for dissecting chromosome fragments with a size of a few hundred nanometers by atomic force microscopy (AFM). By using this method, we demonstrated reproducible dissections of silkworm chromosomes in the pachytene phase. The dissected fragments were successfully recovered on the cantilever tips, as confirmed by fluorescent microscopy using fluorescent stained chromosomes. To recover dissected chromosome fragments from a larger chromosome, such as the human metaphase chromosome of a somatic cell, heat denaturation was found to be effective. Further improvements in this method may lead to a novel tool for isolating valuable genes and/or investigating local genome structures in the near future.