J.A. Veerman

Single molecule mapping of the optical field distribution of probes for near‐field microscopy

The most difficult task in near‐field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear‐force microscopy (both providing geometrical information), by far‐field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near‐field. In this paper, we report on a new method using shear‐force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three‐dimensional optical near‐field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near‐field optical resolution constitutes an order of magnitude improvement over far‐field methods like confocal microscopy.

Tuning fork shear-force feedback

Investigations have been performed on the dynamics of a distance regulation system based on an oscillating probe at resonance. This was examined at a tuning fork shear-force feedback system, which is used as a distance control mechanism in near-field scanning optical microscopy. In this form of microscopy, a tapered optical fiber is attached to the tuning fork and scanned over the sample surface to be imaged. Experiments were performed measuring both amplitude and phase of the oscillation of the tuning fork as a function of driving frequency and tip-sample distance. These experiments reveal that the resonance frequency of the tuning fork changes upon approaching the sample. Both the amplitude and the phase of the tuning fork can be used as distance control parameter in the feedback system. Using the amplitude a second-order behavior is observed, while with phase only a first-order behavior is observed. Numerical calculations confirm these observations. This first-order behavior results in an improved stability of the feedback system. As an example, a sample consisting of DNA strands on mica was imaged which showed the height of the DNA as 1.4 +/- 0.2 nm.

Near-field optical and shear-force microscopy of single fluorophores and DNA molecules

Photodynamics of individual fluorescence molecules has been studied using an aperture-type near-field scanning optical microscope with two channel fluorescence polarisation detection and tuning fork shear-force feedback. The position of maximum fluorescence from individual molecules could be localised with an accuracy of 1 nm. Dynamic processes such as translational and rotational diffusion were observed for molecules adsorbed to a glass surface or embedded in a polymer host. The in-plane molecular dipole orientation could be determined by monitoring the relative contribution of the fluorescence signal in the two perpendicular polarised directions. Rotational dynamics was investigated on 10 ms-1000 s timescale. Shear-force phase feedback was used to obtain topographic imaging of DNA fragments, with a lateral and vertical resolution comparable to scanning force microscopy. A DNA height of 1.4 nm has been measured, an indication of the non-disturbing character of the shear force mechanism.

Individual green fluorescent proteins (GFP) studied by near-field optical microscopy

Since the cloning and subsequent expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria research interest for this protein has increased dramatically. The exclusiveness of GFP as a biological marker derives from the finding that no cofactors are required for the protein’s fluorescence [1]. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be monitored by sensitive fluorescence methods without the need to add external fluorescent dyes. Other advantages of the protein include the possibility of performing genetic manipulation to alter its fluorescent properties, resulting in mutants that exhibit excitation and emission spectra different from those of the wild type (wt)-GFP and in some cases with higher quantum yield [2, 3]. Furthermore, it has been elucidated that the chromophore in the GFP is completely protected from bulk solvent [4]. The high quantum yield of fluorescence observed in ensemble measurements has been attributed to this presumably rigid encapsulation, while the low photodissociation rate observed for GFP has been attributed to the inability of O2 to quench the excited state [4].