ABSTRACT
Chromosomes, DNA, and single fluorescent molecules are studied using an aperture-type near-field scanning optical microscope with tuning fork shear force feedback. Fluorescence in situ hybridization labels on repetitive and single copy probes on human metaphase chromosomes are imaged with a width of 80 nm, allowing their localisation with nanometer accuracy, in direct correlation with the simultaneously obtained topography. Single fluorophores, both in polymer and covalently attached to amino-silanized glass, are imaged using two-channel fluorescence polarization detection. The molecules are selectively excited according to their dipole orientation. The orientation of the dipole moment of all molecules in one image could be directly determined. Rotational dynamics on a 10-ms to 100-s timescale is observed. Finally, shear force imaging of double-stranded DNA with a vertical sensitivity of 0.2 nm is presented. A DNA height of 1.4 nm is measured, which indicates the nondisturbing character of the shear force mechanism.
Subject(s)
Chromosomes, Human, Pair 1/ultrastructure , DNA/ultrastructure , Microscopy/methods , DNA-Binding Proteins/ultrastructure , Fluorescence Polarization , Fluorescent Dyes , Humans , In Situ Hybridization, Fluorescence , Microscopy/instrumentationABSTRACT
Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.
Subject(s)
Chromosomes/chemistry , DNA/analysis , In Situ Hybridization, Fluorescence/methods , Metaphase , Microscopy, Fluorescence , Biotin , Centromere/ultrastructure , Chromosomes/ultrastructure , DNA/genetics , DNA Probes , Digoxigenin , Fluorescein-5-isothiocyanate , Humans , Lymphocytes , Male , Spectrin/genetics , Telomere/ultrastructureABSTRACT
Fluorescence in situ hybridization signals on human metaphase chromosomes are detected by a near-field scanning optical microscope. This makes it possible to localize and identify several fluorescently labeled genomic DNA fragments on a single chromosome with a resolution superior to traditional fluorescence microscopy. Several nucleic acid probes have been used. The hybridization signals are well resolved in the near-field fluorescence images, and the exact location of the probes can be correlated to the topography as it is afforded by the shear-force feedback.
