Micron-sized cargos in living cells are actively transported in opposite directions along microtubules by the molecular motors kinesin and dynein. While these motors have been extensively studied in vitro, the conditions in the cell differ substantially. In particular, drag forces cease to be negligible in vivo. Previous experiments performed in vitro show that opposing loads affect the transport velocity of kinesin and dynein differently, resulting in different force-velocity curves [1, 2]. We have observed evidence for these force-velocity dependences in vivo. First, we quantified the cytoplasmic viscous forces experienced by motors in Drosophila embryos by using a combination of passive microrheology, and a novel approach to active microrheology, with endogenous lipid droplets as probes. We then treated the embryos with inhibitors or promoters of actin polymerization, thus changing the average rheological properties experienced by motor-driven cargo. This allowed us to measure the effect of cytoplasmic drag on the velocity of those same lipid droplets hauled by kinesin and dynein [3, 4]. We find that kinesin and dynein respond differently to cytoplasmic drag forces, with kinesin being load-sensitive at high opposing forces and dynein at low. Our findings agree with and ‐ to our knowledge ‐ constitute the first in vivo validation of the force-velocity curves for kinesin and dynein found in vitro.

Single-molecule function of molecular motors has been well characterized in vitro, but in vivo, motors typically function as part of larger ensembles, including both regulatory proteins and multiple motors. Thus, single-molecule knowledge is only the start of the journey to understand cellular function and organization. The challenge is to use single-molecule information to quantitatively understand transport and its regulation in a cellular context. This article discusses this process focusing on kinesin and dynein, highlighting progress to date, and the challenges involved in using single-molecule function to understand in vivo transport.

The experimental results of the direct measurement of the absolute value of interaction force between the fiber probe of a scanning near-field optical microscope (SNOM) operated in shear force mode and a sample, which were performed using combined SNOM-atomic force microscope setup, are discussed for the out-of-resonance fiber probe excitation mode. We demonstrate that the value of the tapping component of the total force for this mode at typical dither amplitudes is of the order of 10nN and thus is quite comparable with the value of this force for in resonance fiber probe excitation mode. It is also shown that for all modes this force component is essentially smaller than the usually neglected static attraction force, which is of the order of 200nN. The true contact nature of the tip–sample interaction during the out of resonance mode is proven. From this, we conclude that such a detection mode is very promising for operation in liquids, where other modes encounter great difficulties.

Aim of our study is to use Atomic Force Microscopy (AFM) to measure the spring constant for single bovine serum albumin (BSA) – polyclonal antibody to BSA (Ab-BSA) complex (kcomplex) as a function of pulling off force and complex extension. By the combination of AFM operated in the force-spectroscopy mode with the small dithering of the AFM tip we are able to detect the change of the dithering amplitude upon the pulling off process and to derive the value of kcomplex. At the moment of single specific bond rupture the spring constant value of 0.017 ± 0.003 N/m is determined.

Optical properties of microporous polymer membranes coated with metal have been studied using scanning near-field optical microscopy. The electromagnetic field distribution close to pores of various sizes ranging from 35 to 750 nm has been investigated in the different illumination configurations. Individual pores as small as 35 nm were seen in the near field. For directly illuminated membranes the field distribution is related mainly to the pores themselves. However, when surface plasmon polaritons were excited, the field distribution exhibited much more complex structure related to the interaction of surface polaritons with the ensemble of randomly distributed pores and metal surface roughness as well as localized surface plasmon effects. The results will be of importance for the understanding of optical properties of subwavelength apertures in strongly scattering films as well as optics of randomly structured metal films.

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.

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.

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.

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.

Summary form only given. The spatial resolution of scanning near-field optical microscope (SNOM) is limited by the size of an aperture for the light transmission and ranges 50-100 nm, although 20 nm resolution has been demonstrated. Further improvement of the resolution seems problematic for the "classical" SNOM configurations because the number of photons "seeping" through an aperture is rapidly decreasing with the decrease of the aperture size. A number of new approaches to improve the resolution, such as SNOM using fluorescence resonant energy transfer (FRET) between a single fluorescence center of the tip and the sample studied have been proposed.

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.