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

Abstract

An aperture-type near-field optical microscope (NSOM) with two polarization detection channels has been used to image fluorescently labelled DNA with high spatial resolution and single molecule fluorescence sensitivity. The sample has been engineered such that there is only one rhodamine dye per DNA strand. Lateral and vertical DNA dimensions in the shear-force image are 14 ± 2 nm and 1.4 ± 0.2 nm, respectively. No sample deformation was observed under our imaging conditions. Near-field fluorescence imaging of individual fluorophores shows an optical resolution of 70 nm at full-width at half-maximum. Large intensity differences between individual rhodamine molecules attached to DNA are observed from the NSOM images. Statistics on rhodamine dyes in different environments (attached to glass, embedded in a polymer layer and attached to DNA) show bleaching rates of 10 −5 . Total intensity line profiles together with in-plane angle orientation are used to characterize individual dyes. Rhodamine dyes show strong intensity fluctuations independent of the particular environment. These results are in contrast with the more stable photophysical behaviour as observed for carbocyanine molecules embedded in polymer matrices. The mobility of rhodamine-both lateral and rotational-is clearly influenced by its immediate surrounding and attachment to the surface.

Near-Field Scanning Optical Microscopy of Single Fluorescent Dendritic Molecules

“…Single-molecule rotational movements have been previously observed for, e.g., dye-DNA complexes linked to a silica substrate and within dye-doped polymer systems. 4,10,20 For the dye-DNA complexes, rotational movements were attributed to the flexible DNA-linking between dye and surface. Orientational jumps in polymer environments have been related to lowfrequency polymer side-group motions.…”

Near-Field Scanning Optical Microscopy of Single Fluorescent Dendritic Molecules

“…Single-molecule rotational movements have been previously observed for, e.g., dye-DNA complexes linked to a silica substrate and within dye-doped polymer systems. 4,10,20 For the dye-DNA complexes, rotational movements were attributed to the flexible DNA-linking between dye and surface. Orientational jumps in polymer environments have been related to lowfrequency polymer side-group motions.…”

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

“…In this process, the high-resolution surface topography of cells is simultaneously generated by an additional shear-force feedback signal, which is applied to keep the probe in the near-field region of the cell sample. Through this method, NSOM is particularly suitable for investigating single molecule locations with high optical resolution, 50 as well as the distribution of membrane components on the cell surface with precise membrane structures at the nanoscale. 51 As expected, the ability of NSOM to map and detect co-localized proteins in cell membranes with a super-resolution of 100 nm was demonstrated, 52 and proved that NSOM could be specifically applied to the immuno-fluorescence studies of biomolecules on the cell surface, as well as highlighting the possibility and applicability of NSOM for the in situ single molecule imaging of cell membranes.…”

Nanophotonic approaches for nanoscale imaging and single‐molecule detection at ultrahigh concentrations

“…Since light of different wavelengths can be simultaneously funneled through the same subwavelength aperture, NSOM allows simultaneous multicolor excitation free from chromatic aberrations (de Bakker et al, ; Enderle et al, ). In addition, the small excitation volume (10 5 vs. 10 8 nm 3 as obtained in confocal microscopy) reduces dramatically the cytoplasm background fluorescence, enabling multicolor single‐molecule detection with high signal‐to‐background ratios (de Lange et al, ; Dûfrene and Garcia‐Parajo, ; Garcia‐Parajo et al, ; Hinterdorfer et al, ; van Zanten et al, ) and localization accuracies (i.e., determination of the center‐of‐mass position of the nanoscopy fluorescent spot) better than 3 nm on intact cell membranes under physiological conditions (van Zanten et al, ).…”

Near-Field Scanning Optical Microscopy of Single Fluorescent Dendritic Molecules

“…Single-molecule rotational movements have been previously observed for, e.g., dye-DNA complexes linked to a silica substrate and within dye-doped polymer systems. 4,10,20 For the dye-DNA complexes, rotational movements were attributed to the flexible DNA-linking between dye and surface. Orientational jumps in polymer environments have been related to lowfrequency polymer side-group motions.…”

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

“…In this process, the high-resolution surface topography of cells is simultaneously generated by an additional shear-force feedback signal, which is applied to keep the probe in the near-field region of the cell sample. Through this method, NSOM is particularly suitable for investigating single molecule locations with high optical resolution, 50 as well as the distribution of membrane components on the cell surface with precise membrane structures at the nanoscale. 51 As expected, the ability of NSOM to map and detect co-localized proteins in cell membranes with a super-resolution of 100 nm was demonstrated, 52 and proved that NSOM could be specifically applied to the immuno-fluorescence studies of biomolecules on the cell surface, as well as highlighting the possibility and applicability of NSOM for the in situ single molecule imaging of cell membranes.…”

Nanophotonic approaches for nanoscale imaging and single‐molecule detection at ultrahigh concentrations

“…Since light of different wavelengths can be simultaneously funneled through the same subwavelength aperture, NSOM allows simultaneous multicolor excitation free from chromatic aberrations (de Bakker et al, ; Enderle et al, ). In addition, the small excitation volume (10 5 vs. 10 8 nm 3 as obtained in confocal microscopy) reduces dramatically the cytoplasm background fluorescence, enabling multicolor single‐molecule detection with high signal‐to‐background ratios (de Lange et al, ; Dûfrene and Garcia‐Parajo, ; Garcia‐Parajo et al, ; Hinterdorfer et al, ; van Zanten et al, ) and localization accuracies (i.e., determination of the center‐of‐mass position of the nanoscopy fluorescent spot) better than 3 nm on intact cell membranes under physiological conditions (van Zanten et al, ).…”

Near-Field Scanning Optical Microscopy of Single Fluorescent Dendritic Molecules

“…Single-molecule rotational movements have been previously observed for, e.g., dye-DNA complexes linked to a silica substrate and within dye-doped polymer systems. 4,10,20 For the dye-DNA complexes, rotational movements were attributed to the flexible DNA-linking between dye and surface. Orientational jumps in polymer environments have been related to lowfrequency polymer side-group motions.…”

In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

“…In this process, the high-resolution surface topography of cells is simultaneously generated by an additional shear-force feedback signal, which is applied to keep the probe in the near-field region of the cell sample. Through this method, NSOM is particularly suitable for investigating single molecule locations with high optical resolution, 50 as well as the distribution of membrane components on the cell surface with precise membrane structures at the nanoscale. 51 As expected, the ability of NSOM to map and detect co-localized proteins in cell membranes with a super-resolution of 100 nm was demonstrated, 52 and proved that NSOM could be specifically applied to the immuno-fluorescence studies of biomolecules on the cell surface, as well as highlighting the possibility and applicability of NSOM for the in situ single molecule imaging of cell membranes.…”

Nanophotonic approaches for nanoscale imaging and single‐molecule detection at ultrahigh concentrations

“…Since light of different wavelengths can be simultaneously funneled through the same subwavelength aperture, NSOM allows simultaneous multicolor excitation free from chromatic aberrations (de Bakker et al, ; Enderle et al, ). In addition, the small excitation volume (10 5 vs. 10 8 nm 3 as obtained in confocal microscopy) reduces dramatically the cytoplasm background fluorescence, enabling multicolor single‐molecule detection with high signal‐to‐background ratios (de Lange et al, ; Dûfrene and Garcia‐Parajo, ; Garcia‐Parajo et al, ; Hinterdorfer et al, ; van Zanten et al, ) and localization accuracies (i.e., determination of the center‐of‐mass position of the nanoscopy fluorescent spot) better than 3 nm on intact cell membranes under physiological conditions (van Zanten et al, ).…”