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.

Analysis of individual (macro)molecules and proteins using near-field optics

“…Alternatively they can be incomplete or nonfully folded GFPs, which will appear on the topographic image but are nonfluorescent. 23,59 Finally, the proteins can be in a temporary nonemissive state during the time of imaging. Upon sequential imaging of the same area we find different proteins lightning up from image to image.…”

“…Single molecule ͑center-of-mass͒ translational motions have been studied on fluid lipid membranes 14 in polymer matrices, [15][16][17] along actin filaments, 18,19 and DNA. 20 Similarly orientational mobility 17,[21][22][23] has been observed on a typical timescale of milliseconds to tens of seconds. Such experiments are useful to probe polymer mobility, membrane transport mechanisms, and dynamic interactions of macromolecules like proteins and DNA.…”

“…Thus optical and topographic molecular information are obtained simultaneously, which enables direct correlation of fluorescent and structural properties. 23,[51][52][53][54] Finally, the scanning near-field probe can be used to actively manipulate and control the sample by e.g., a local electric field 55 or energy transfer. 56 The first near-field optical observation of single molecule fluorescence in 1993 by Betzig and Chichester 1 stimulated the field of single molecule detection but also set a standard in near-field optics, especially with their 3Dorientation determination of single molecules.…”

“…56 The first near-field optical observation of single molecule fluorescence in 1993 by Betzig and Chichester 1 stimulated the field of single molecule detection but also set a standard in near-field optics, especially with their 3Dorientation determination of single molecules. Subsequent near-field experiments involved single molecule spectra 24 and fluorescence lifetimes, 27,28 lateral diffusion and rotation in a polymer host, 15,17 single-pair FRET, 57 single fluorophore labeled DNA strands, 23,58 and green fluorescent proteins. 59 Yet, despite the potential of near-field optics, today almost all single-molecule fluorescence experiments are performed using far-field microscopy because of the much less complexity.…”

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, ).…”