Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0-100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.     

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.     

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.     

Near-field effects in single molecule emission

We present the first experimental proof of the influence of a nearby nano-sized metal object on the angular photon emission by a single molecule. A novel angular sensitive detection scheme is implemented in an existing near-field scanning optical microscope (NSOM). The positioning accuracy (∼1 nm) of the NSOM allows a systematic investigation of the intensity ratio between two different half-spaces as a function of the position of the metal–glass interfaces of the probe with respect to the single emitter. The observed effects are shown to be particularly strong for molecules that are excited mainly below the rims of the aperture. An excellent agreement is found between experiments and numerical simulations for these molecules. The observed angular redistribution of the angular emission of a single molecule could explain the alteration of the emission polarization observed for certain molecules in earlier experiments (Veerman et al. (1999) J. Microsc. 194 , 477–482).     

Single beam optical tweezers setup with backscattered light detection for three-dimensional measurements on DNA and nanopores

We introduce a versatile and high precision three-dimensional optical tweezers setup with minimal optical interference to measure small forces and manipulate single molecules in the vicinity of a weak reflective surface. Our tweezers system integrates an inverted optical microscope with a single IR-laser beam that is spatially filtered in an appropriate way to allow force measurements in three dimensions with remarkably high precision when operated in backscattered light detection mode. The setup was tested by overstretching a lambda-DNA in x and z directions (perpendicular and along the optical axis), and by manipulating individual lambda-DNA molecules in the vicinity of a nanopore that allowed quantitative single molecule threading experiments with minimal optical interference.     

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.     

Micromanipulation of chloroplasts using optical tweezers

This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two-point contact on a single chloroplast or two-point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles.  The study also confirms that the chloroplasts are very thin and extremely cup-shaped with a concave inner surface and a convex outer surface.     

The tetrahedral tip as a probe for scanning near-field optical microscopy at 30 nm resolution

The tetrahedral tip is introduced as a new type of a probe for scanning near-field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30 nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30 nm.     

Near-field fluorescence imaging of single molecules with a resolution in the range of 10 nm

We demonstrate fluorescence imaging of single molecules, by near-field scanning optical microscopy (NSOM), using the illumination-collection mode of operation, with an aperture probe. Fluorescence images of single dye molecules were obtained with a spatial resolution of 15 nm, which is smaller than the diameter of the aperture (20 nm) of the probe employed. Such super-resolution may be attributable to non-radiative energy transfer from the molecules to the coated metal of the probe since the resolution obtained in the case of conventional NSOM is limited to 30–50 nm due to penetration of light into the metal.     

Shear force imaging of DNA in a near-field scanning optical microscope (NSOM)

A near-field scanning optical module has been constructed as an accessory for a Nanoscope IIIa commercial scanning probe microscope. Distance feedback and topographic registration are accomplished with an uncoated optical fibre scanning tip by implementation of the shear force technique. The tip is driven by a piezoelectric actuator at a resonance frequency of 8–80 kHz. A laser diode beam is scattered by the tip and detected by a split photodiode, with lock-in detection of the difference signal. The amplitude ( r ) and phase (τ) responses were characterized as a function of the calibrated tip–sample separation. Using an r cos τ feedback signal, imaging of pUC18 relaxed circular plasmid DNA spread on mica precoated with cetylpyridinium chloride was achieved. The apparent width (28 ± 5 nm) was approximately four times that achieved by scanning force measurements with the same instrument; the apparent height of the DNA (0.6 ± 0.3 nm) was similar with the two techniques. These results demonstrate the applicability of the shear force signal for imaging biological macromolecules according to topography and in conjunction with the optical signals of a near-field scanning optical microscope (NSOM).     

Kinetics of TmHU binding to DNA as observed by optical tweezers

The kinetics of binding for the histone-like protein TmHU (from Thermotoga maritima) to DNA is analyzed on a single molecule level by use of optical tweezers. For the reaction rate a pronounced concentration-dependence is found with an "all or nothing"-limit which suggests the cooperative nature of the binding-reaction. By analyzing the statistics of mechanically induced dissociation-events of TmHU from DNA multiple reaction sites are observed to become more likely with increasing TmHU concentration. This is interpreted as a hint for a secondary organizational level of the TmHU/DNA complex. The reaction rate of TmHU binding to DNA is remarkably higher than that of the HU protein from Escherichia coli which will be discussed.     

Near‐field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores

We have coupled a spectrophotometer with a scanning near‐field optical microscope to obtain, with a single scan, simultaneously scanning near‐field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.     

Evaluation of nano-optical probe from scanning near-field optical microscope images

We studied a nanometre-sized optical probe in a scanning near-field optical microscope. The probe profile is determined by using a knife-edge method and a modulated transfer function evaluation method which uses nanometre-sized line-and-space tungsten patterns (with spaces 1 microm to 50 nm apart) on SiO2 substrates. The aluminium-covered, pipette-pulled fibre probe used here has two optical probes: one with a large diameter (350 nm) and the other with a small diameter (10 nm). The small-diameter probe has an optical intensity approximately 63 times larger than that of the large-diameter probe, but the power is about 1/25 of that of the large probe.     

Note: Direct force and ionic-current measurements on DNA in a nanocapillary

We have developed optical tweezers, with force measurements based on fast video tracking, for analysis and control of DNA translocation through nanocapillaries. Nanocapillaries are single-molecule biosensors with very similar characteristics to solid-state nanopores. Our novel experimental setup allows for ionic-current measurements in which the nanocapillary is oriented perpendicular to the trapping laser. Using video-based particle tracking, we are able to measure the position of DNA coated colloids at sub-millisecond resolution and in real-time. We present the first electrophoretic force and simultaneous ionic-current measurements of a single DNA molecule inside the orifice of a nanocapillary.     

Evaluation of nano-optical probe from scanning near-field optical microscope images

We studied a nanometre-sized optical probe in a scanning near-field optical microscope. The probe profile is determined by using a knife-edge method and a modulated transfer function evaluation method which uses nanometre-sized line-and-space tungsten patterns (with spaces 1 μm to 50 nm apart) on SiO₂ substrates. The aluminium-covered, pipette-pulled fibre probe used here has two optical probes: one with a large diameter (350 nm) and the other with a small diameter (10 nm). The small-diameter probe has an optical intensity ≈63 times larger than that of the large-diameter probe, but the power is about 1/25 of that of the large probe.     

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy

A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers

A Nd‐YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament‐dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR‐laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 µm but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament‐mediated processes.     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.     

Are electron tweezers possible?

Positively answering the question in the title, we demonstrate in this work single electron beam trapping and steering of 20–300 nm solid Al nanoparticles generated inside opaque submicron-sized molten Al–Si eutectic alloy spheres. Imaging of solid nanoparticles and liquid alloy in real time was performed using energy filtering in an analytical transmission electron microscope (TEM). Energy-filtering TEM combined with valence electron energy-loss spectroscopy enabled us to investigate in situ nanoscale transformations of the internal structure, temperature dependence of plasmon losses, and local electronic and optical properties under melting and crystallization of individual binary alloy particles. For particles below 20 nm in size, enhanced vibrations of the dynamic solid–liquid interface due to instabilities near the critical threshold were observed just before melting. The obtained results indicate that focused electron beams can act as a tool for manipulation of metal nanoparticles by transferring linear and angular mechanical momenta. Such thermally assisted electron tweezers can be utilized for touchless manipulation and processing of individual nano-objects and potentially for fabrication of assembled nanodevices with atomic level sensitivity and lateral resolution provided by modern electron optical systems. This is by three orders of magnitude better than for light microscopy utilized in conventional optical tweezers. New research directions and potential applications of trapping and tracking of nano-objects by focused electron beams are outlined.