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

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.

Near‐field optical microscopy for DNA studies at the single molecular level

“…The samples were prepared by embedding the dyes in PMMA as described elsewhere [21]. Similarly, as a reference calibration sample, carbocyanine dye molecules (DiI-C 18 from Molecular Probes, D-282) were also investigated.…”

Interaction of an Asymmetric Scanning Near Field Optical Microscopy Probe With Fluorescent Molecules

“…SNOM looks particularly suitable for this purpose due to the strong localization of the field emitted by the probe; moreover, due to the presence of transverse and longitudinal field components close to the probe apex, it has been used for the determination of the three-dimensional (3D) orientation of single fluorophores by controlling the polarization of the excitation light [29]. Aperture SNOM probes have been employed for fluorescence studies on biological molecules and polymers [24,[28][29][30][31].…”

Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy

“…This is especially helpful for biological applications that often exhibit complex morphologies. [20][21][22][23][24][25] While technically difficult due to the strict requirements on tip quality, single molecule NSOM orientation measurements offer a new perspective on sample organization. These measurements are widely applicable for samples ranging from highly ordered crystals to highly disordered polymers or glass systems.…”