John Bechhoefer

Calibration of atomic‐force microscope tips

Images and force measurements taken by an atomic‐force microscope (AFM) depend greatly on the properties of the spring and tip used to probe the sample’s surface. In this article, we describe a simple, nondestructive procedure for measuring the force constant, resonant frequency, and quality factor of an AFM cantilever spring and the effective radius of curvature of an AFM tip. Our procedure uses the AFM itself and does not require additional equipment.

High-Precision Test of Landauer's Principle in a Feedback Trap

We confirm Landauers 1961 hypothesis that reducing the number of possible macroscopic states in a system by a factor of 2 requires work of at least kTln2. Our experiment uses a colloidal particle in a time-dependent, virtual potential created by a feedback trap to implement Landauers erasure operation. In a control experiment, similar manipulations that do not reduce the number of system states can be done reversibly. Erasing information thus requires work. In individual cycles, the work to erase can be below the Landauer limit, consistent with the Jarzynski equality.

Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing

Microarrays are powerful tools to probe genome‐wide replication kinetics. The rich data sets that result contain more information than has been extracted by current methods of analysis. In this paper, we present an analytical model that incorporates probabilistic initiation of origins and passive replication. Using the model, we performed least‐squares fits to a set of recently published time course microarray data on Saccharomyces cerevisiae. We extracted the distribution of firing times for each origin and found that the later an origin fires on average, the greater the variation in firing times. To explain this trend, we propose a model where earlier‐firing origins have more initiator complexes loaded and a more accessible chromatin environment. The model demonstrates how initiation can be stochastic and yet occur at defined times during S phase, without an explicit timing program. Furthermore, we hypothesize that the initiators in this model correspond to loaded minichromosome maintenance complexes. This model is the first to suggest a detailed, testable, biochemically plausible mechanism for the regulation of replication timing in eukaryotes.

Kinetic model of DNA replication in eukaryotic organisms

We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.

Erratum: ‘‘Calibration of atomic‐force microscope tips’’ [Rev. Sci. Instrum. 64, 1868 (1993)]

In our calibration of atomic-force microscope cantilevers, we neglected to correct for the frequency response of the optical-detection electronics. The response to cantilever vibrations will have a high-frequency cut-off, which, in our case, was higher than the resonant frequency of the cantilever. Our results were not affected, but for higher resonant frequencies, one should calibrate the detector response. We thank V. Croquette for raising this point.

The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

Replication timing and its emergence from stochastic processes

The temporal organization of DNA replication has puzzled cell biologists since before the mechanism of replication was understood. The realization that replication timing correlates with important features, such as transcription, chromatin structure and genome evolution, and is misregulated in cancer and aging has only deepened the fascination. Many ideas about replication timing have been proposed, but most have been short on mechanistic detail. However, recent work has begun to elucidate basic principles of replication timing. In particular, mathematical modeling of replication kinetics in several systems has shown that the reproducible replication timing patterns seen in population studies can be explained by stochastic origin firing at the single-cell level. This work suggests that replication timing need not be controlled by a hierarchical mechanism that imposes replication timing from a central regulator, but instead results from simple rules that affect individual origins.

An experimental study of the onset of parametrically pumped surface waves in viscous fluids

We measure the threshold accelerations necessary to excite surface waves in a vertically vibrated fluid container (the Faraday instability). Under the proper conditions, the thresholds and onset wavelengths agree with recent theoretical predictions for a laterally infinite, finite-depth container filled with a viscous fluid. Experimentally, we show that by using a viscous, non-polar fluid, the finite-size effects of sidewalls and the effects of surface contamination can be made negligible. We also show that finite-size corrections are of order h/L , where h is the fluid depth and L the container size. Based on these measurements, one can more easily interpret certain unexpected observations from previous experimental studies of the Faraday instability.

Manipulation of van der Waals forces to improve image resolution in atomic‐force microscopy

Although the atomic force microscope (AFM) resembles superficially the scanning tunneling microscope (STM), its imaging resolution is in general much coarser. For the AFM, long‐range interactions—most notably the van der Waals force—imply that image resolution is set by the macroscopic tip radius rather than by a single atom, as with the STM. Experimentally, we show that van der Waals forces can be measured using an AFM. By immersing tip and sample in an appropriate fluid, we can effectively eliminate the van der Waals force, leading to a marked improvement in AFM image quality.

Measurement and manipulation of van der Waals forces in atomic‐force microscopy

An understanding of the interaction between tip and sample in atomic‐force microscopy is needed to interpret atomic‐force‐microscope (AFM) images. In contact mode, the strength of the van der Waals (vdW) force sets image resolution; in noncontact mode, local gradients in the vdW force are imaged. By immersing tip and sample in an appropriate fluid, we can decrease the vdW forces and even change their sign. Selecting a fluid that leads to a small repulsive vdW force can greatly improve image resolution and eliminates problems caused by the well‐known tip‐snapping instability. To measure the vdW interactions produced by different fluids, we have developed ways to calibrate the spring constant and sharpness of AFM tips and to measure accurately the Hamaker constant of vdW interactions. These techniques show that the AFM can be used for local force measurements with an accuracy approaching that of surface‐force apparatuses. As an example, we have observed the crossover from nonretarded to retarded vdW forces ...