Koen Visscher

Single kinesin molecules studied with a molecular force clamp.

Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis–Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

Force production by single kinesin motors

Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis–Menten relationship.

Construction of multiple-beam optical traps with nanometer-resolution position sensing

We describe the design and construction of two different types of multiple-beam optical tweezers, each equipped with nanometer-resolution position detectors. Multiple optical traps can be created either by splitting a laser beam in two parts, based on its polarization, or time-sharing a single beam among several different locations. The advantages and disadvantages of optical tweezers based on either scheme are discussed, along with details of specific implementations. Various ways to detect microscopic movements of an optically trapped object are presented and compared, including designs that are relatively insensitive to absolute location of a trapped particle within the field of view. Two of many possible applications for such instruments are illustrated: the detection of molecular steps by kinesin motor molecules, and determinations of the stiffness of single microtubules.

Confocal imaging with bilateral scanning and array detectors

A novel arrangement for confocal microscopy is presented, in which the key elements are the use of an array detector such as a CCD for confocal image collection and the use of one double‐sided scanning mirror element for bilaterally scanning the object and collecting the data on the CCD. The resulting arrangement is shown to be capable of confocal imaging with high photon efficiency under adjustable conditions of confocality and varying image acquisition rates, i.e. from slow speed up to real‐time imaging. Either laser or conventional light sources may be utilized. In addition to CCD registration, direct observation by eye of the confocal image in fluorescence is also possible.

An objective, model-independent method for detection of non-uniform steps in noisy signals

Biophysical techniques, such as single molecule FRET, fluorescence microscopy, single ion-channel patch clamping, and optical tweezers often yield data that are noisy time series containing discrete steps. Here we present a method enabling objective identification of nonuniform steps present in such noisy data. Our method does not require the assumption of any underlying kinetic or state models and is thus particularly useful for analysis of novel and poorly understood systems. In contrast to other model-independent methods, no parameters or other information is taken from the user. We find that, at high noise levels, our method exceeds the performance of other model-independent methods in accurately locating steps in simulated noisy data.

Interaction of the HIV-1 frameshift signal with the ribosome

Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.

Imaging modes for bilateral confocal scanning microscopy

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two‐dimensional (2‐D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow‐type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non‐confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time‐resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2‐D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real‐time or can use a cooled CCD at (very) low light level for image integration over an extended period.

Fluorescence bleach rate imaging

Bleach rate imaging on a (cooled) CCD can be easily achieved using a confocal microscope with bilateral scanning and detection coupled to a workstation; it is as easy as acquiring regular fluorescence images. Several analysis and display methods for bleach rate imaging are presented such as the bleach map (and its inverse) and a matrix‐based decomposition method for multi‐labelled specimens based on the bleach rate differences between the dyes used. With these tools, bleach‐rate‐based imaging can become a viable alternative to multiple labelling techniques for component identification in fluorescent specimens.

Colloidal epitaxy: Playing with the boundary conditions of colloidal crystallization

We have studied, with quantitative confocal microscopy, epitaxial colloidal crystal growth of particles interacting with an almost hard-sphere (HS) potential in a gravitational field and density matched colloids interacting with a long-range (LR) repulsive potential with a body-centred cubic (BCC) equilibrium crystal phase. We show that in both cases it is possible to grow thick, stacking fault-free metastable crystals: close-packed crystals with any stacking sequence, including hexagonal close packed (HCP), for the HS particles and face-centred cubic (FCC) in the case of the LR colloids. In accordance with recent computer simulations done for HS particles it was found that the optimal lattice constant to grow HS HCP crystals was larger than that of equilibrium FCC crystals. In addition, because of the absence of gravity, pre-freezing could be observed for the particles with the LR potential on a template of charged lines. We also argue that the ability to manipulate colloids with highly focused light, optical traps or tweezers, will become an important tool in both the study of colloidal crystallization and in making new structures. We show how cheap 2D and 3D templates can be made with optical tweezers and demonstrate, in proof of principle experiments with core-shell colloids, how light fields can generate crystal nuclei and other structures in the bulk of concentrated dispersions and how the effect of these structures on the rest of a dispersion can be studied quantitatively in 3D.

Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing

A confocal microscope can be considered as a 3D sampling instrument for collecting data from spatial structures, especially biological ones. Optimal data collection in confocal microscopes requires the adaptation of the dimensions of the sampling volume to the lateral and axial raster parameters employed during data collection. It is shown how, in principle, the collection volume can be partly manipulated by the use of variable pinholes both in the illumination and detection paths. The effective confocal spot will depend on the optics used, the degree of aberration, and the alignment of the instrument. Measurements of the axial response both in fluorescence and reflection for some high N.A. lens systems as a function of the above factors are presented. The use of variable pinholes in computer-controlled instruments is discussed, especially in relation to operation in fluorescence. It is indicated that proper interpretation and processing of 3D confocal data requires at least approximate knowledge of the applicable 3D response function.