G. T. Shubeita

Force spectroscopy with a small dithering of AFM tip: A method of direct and continuous measurement of the spring constant of single molecules and molecular complexes

A new method of direct and continuous measurement of the spring constant of single molecule or molecular complex is elaborated. To that end the standard force spectroscopy technique with functionalized tips and samples is combined with a small dithering of the tip. The change of the dithering amplitude as a function of the pulling force is measured to extract the spring constant of the complex. The potentialities of this method are illustrated for the experiments with single bovine serum albumin-its polyclonal antibody (Ab-BSA) and fibrinogen-fibrinogen complexes.

Scanning near‐field optical microscopy using semiconductor nanocrystals as a local fluorescence and fluorescence resonance energy transfer source

Local fluorescence probes based on CdSe semiconductor nanocrystals were prepared and tested by recording scanning near‐field optical microscopy (SNOM) images of calibration samples and fluorescence resonance energy transfer SNOM (FRET SNOM) images of acceptor dye molecules inhomogeneously deposited onto a glass substrate. Thousands of nanocrystals contribute to the signal when this probe is used as a local fluorescence source while only tens of those (the most apical) are involved in imaging for the FRET SNOM operation mode. The dip‐coating method used to make the probe enables diminishing the number of active fluorescent nanocrystals easily. Prospects to realize FRET SNOM based on a single fluorescence centre using such an approach are briefly described.

Local fluorescent probes for the fluorescence resonance energy transfer scanning near-field optical microscopy

We present fluorescence resonance energy transfer (FRET) images of donor dye molecule clusters recorded using a local fluorescence probe for scanning near-field optical microscopy (SNOM): standard apertured SNOM fiber tip coated with the 30–100-nm-thick polymer layer stained with the acceptor dye molecules. The tip works as a “self-sharpening pencil”: the apical layers of the FRET-active tip coating are mechanically worn out during scanning thus continuously exposing a fresh active apex to continue imaging. Only a few tens of acceptor molecules are used to form the optical images, and using such an approach spatial resolution better than the size of the aperture is achievable.

INVESTIGATION OF NANOLOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER FOR SCANNING PROBE MICROSCOPY

Fluorescence resonance energy transfer (FRET) has been observed between donor dye molecules deposited onto the sample surface and acceptor dye molecules deposited onto the scanning near-field optical microscope (SNOM) or atomic force microscope tip. FRET was observed only when the tip acquired a contact with the sample and took place in a region of few tens of square nanometers in size when thousands (hundreds) of molecules are involved. In view of the obtained results, the perspectives for the construction of a one-atom FRET SNOM are described.

Time-gated scanning near-field optical microscopy

A time-gated scanning near-field optical microscope (SNOM) has been developed. The optical signal was recorded at the precise moment during the fiber tip oscillation period when it made contact with the sample surface. The use of such an approach substantially improves the signal-to-noise ratio for common SNOM applications such as frustrated total internal reflection, surface plasmon imaging, and fluorescence resonance energy transfer-based SNOM. The observed dependence of the frustrated total internal reflection optical signal on the gate delay time confirms that repetitive bumping is the mechanism responsible for the shear force tip–sample interaction.

Scanning near-field optical microscopy based on the heterodyne phase-controlled oscillator method

The heterodyne phase-controlled oscillator method to monitor the resonance frequency and quality factor of the tip oscillations was used to control the scanning near-field optical microscope (SNOM) and to study the nature of the shear-force interaction routinely used in SNOM. Both optical and nonoptical (tuning fork-based) detection schemes of the shear force have been investigated using the same electronic unit, which enables a direct comparison of the results. It is shown that the possibility to record simultaneously the topography and dissipative interaction (Q-factor) channels gives additional information about the sample and helps to interpret the data in a manner analogous to that of a usual dynamic force microscope. The peculiarities of the recorded approach curves (increase of the resonance frequency and Q factor when the tip approaches the sample) are consistent with the “repetitive bumping” mechanism of tip–sample interaction for the shear force. Evidence for the transition from the bumping to t...

Towards the Fluorescence Resonance Energy Transfer (FRET) Scanning Near-Field Optical Microscopy: Investigation of Nanolocal FRET Processes and FRET Probe Microscope ¶

The fluorescence resonance energy-transfer (FRET) process is investigated between donor dye molecules deposited on the sample surface and acceptor dye molecules deposited on the tips of scanning near-field and atomic force microscopes. The FRET process was observed only when the tip acquired contact with the sample and took place in regions of sizes of only a few tens of nanometers with only a few thousands (or even hundreds) of molecules involved. The dependence of the FRET intensity on the tip-sample acting force is recorded and interpreted. In relation to the obtained results, the construction of a previously proposed one-atom FRET SNOM is described.

Characterization of atomic force microscope probes at low temperatures

Different types of atomic force microscopy (AFM) probes were characterized under ultrahigh vacuum conditions and at low temperatures. Properties of AFM probes, such as the resonance frequency, the spring constant and quality factor of cantilevers, depend on temperature. A typical shift in the resonance frequency as a function of temperature was observed for all kinds of cantilevers studied. This was related to the change in temperature of Young’s modulus of the cantilever material. Moreover, force–distance curves acquired at low temperatures and on different substrates, elucidate the importance of the hydrophobicity of the sample surface and that of the tips for lowering adhesion forces. Finally, all of the probes were imaged in a scanning electron microscope as a function of the temperature. A bending of the coated cantilever at low temperatures was observed, which explains the peculiar force–distance curves. As a consequence, the use of uncoated cantilevers for low-temperature applications is recommended.

Direct measurement of the absolute value of the interaction force between the fiber probe and the sample in a scanning near-field optical microscope

The absolute values of the force exerted by the fiber probe of a scanning near-field optical microscope onto the surface were measured using an atomic force microscope in ambient conditions. We demonstrate that a usually neglected static attraction force is dominant at small dither amplitudes and is of the order of 200 nN. The tapping component of the force, often referred to as shear force, is of the order of 1 nN at these conditions for both the tuning fork-based and optical in resonance detection schemes. Other peculiarities of the shear force interaction are also discussed.

On the possibility of observation of single quadrupoles by fluorescence resonance energy transfer scanning near-field optical microscopy

Abstract The possibility to observe single quadrupoles by the recently proposed fluorescence resonance energy transfer scanning near-field optical microscopy (FRET SNOM) is analyzed. When an excited dipole donor center of the SNOM tip is scanned close to a quadrupole molecule of the sample (at a distance of 1–2 nm), the probability of donor-quadrupole FRET becomes close to unity. Thus a single quadrupole can be imaged as a donor fluorescence quenching or by observing dielectric tip-enhanced fluorescence of the quadrupole. To the best of our knowledge, this is the only existing possibility to visualize single quadrupoles.