Near-Field Fluorescence Imaging of Genetic Material: Toward the Molecular Limit

N.F. van Hulst, M.F. Garcia Parajo, M.H.P. Moers, J.A. Veerman, A.G.T. Ruiter

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Chromosomes, DNA, and single fluorescent molecules are studied using an aperture-type near-held 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 localization 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.
Original languageEnglish
Pages (from-to)222-231
Number of pages10
JournalJournal of structural biology
Issue number2
Publication statusPublished - 1997


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