Experimental probes

Quantitative 3D imaging of whole, unstained cells
Huaidong Jiang, Changyong Song, Chien-Chun Chen, Rui Xu, Kevin S. Raines, Benjamin P. Fahimian, Chien-Hung Lu, Ting-Kuo Lee, Akio Nakashima, Jun Urano, Tetsuya Ishikawa, Fuyuhiko Tamanoi, Jianwei Miao
Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantita- tive 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3–5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50–60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5–10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.
JPEGs: Quantitative 3D imaging of whole, unstained cells.jpg (466.93 KB)
PDFs: PNAS-2010-Jiang-1000156107.pdf (13.53 MB)
Research categories: Experimental probes

Electron tomography at 2.4 Å resolution
M. C. Scott, Chien-Chun Chen, Matthew Mecklenburg, Chun Zhu, Rui Xu, Peter Ercius, Ulrich Dahmen, B. C. Regan & Jianwei Miao
Transmission electron microscopy (TEM) is a powerful imaging tool that has found broad application in materials science, nanoscience and biology. With the introduction of aberration-corrected electron lenses, both the spatial resolution and image quality in TEM have been significantly improved and resolution below 0.5 Å has been demonstrated. To reveal the 3D structure of thin samples, electron tomography is the method of choice, with resolutions of ~1 nm currently achievable. Recently, discrete tomography has been used to generate a 3D atomic reconstruction of a silver nanoparticle 2-3 nm in diameter, but this statistical method assumes prior knowledge of the particle’s lattice structure and requires that the atoms fit rigidly on that lattice. Here we report the experimental demonstration of a general electron tomography method that achieves atomic scale resolution without initial assumptions about the sample structure. By combining a novel projection alignment and tomographic reconstruction method with scanning transmission electron microscopy, we have determined the 3D structure of a ~10 nm gold nanoparticle at 2.4 Å resolution. While we cannot definitively locate all of the atoms inside the nanoparticle, individual atoms are observed in some regions of the particle and several grains are identified at three dimensions. The 3D surface morphology and internal lattice structure revealed are consistent with a distorted icosahedral multiply-twinned particle. We anticipate that this general method can be applied not only to determine the 3D structure of nanomaterials at atomic scale resolution, but also to improve the spatial resolution and image quality in other tomography fields.
JPEGs: Electron tomography at 2.4 Å resolution.jpg (1.15 MB)
PDFs: 1108.5350v4.pdf (21.47 MB)
Research categories: Experimental probes

Probing single cells using flow in microfluidics
Probing single cells using flow in microfluidics,  Qi D*, Hoelzle DJ*, Rowat AC (2012) Eur. Phys. J., 204: 85-101. *equal contribution
Enabling fluids to be manipulated on the micron-scale, microfluidic technologies have facilitated major advances in how we study cells. In this review, we highlight key developments in how flow in microfluidic devices is exploited to investigate the behavior of individual cells, from trapping and positioning single cells to probing cell deformability. Exploiting the properties of fluids and flow patterns in microchannels makes it possible to study large populations of single cells at micron-length scales with increased throughput and efficiency.
PDFs: Probing single cells.pdf
Research categories: Experimental probes

The complete bending energy function for nicked DNA
Hao Qu and Giovanni Zocchi, “The complete bending energy function for nicked DNA”, Europhys. Lett. 94, 18003 (2011).
We derive an analytic expression for the bending elastic energy of short DNA molecules, valid in the entire range from low to high energies. The elastic energy depends on three parameters: the length of the molecule (2L), the bending modulus B, and a critical torque τc at which the molecule develops a kink. In the kinked state, the elastic energy is linear in the kink angle, i.e. the torque at the kink is constant (= τc ). τc depends (weakly) on the sequence around the nick, but is about 27 pN × nm. We measure it for a specific sequence, through experiments where the elastic energy of constrained DNA molecules is directly measured.
JPEGs: DNA_stressed.jpg (733.84 KB)
PDFs: Hao_2_reprint.pdf (7.82 MB)
Research categories: Biological Macromolecules, Experimental probes

Mechanisms of chiral discrimination by topoisomerase IV
K. C. Neuman, G. Charvin, D. Bensimon, and V. Croquette
Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.
JPEGs: Mechanisms of chiral discrimination.jpg (500.38 KB)
PDFs: Mechanisms of chiral discrimination.pdf (4.31 MB)
Research categories: Biological Macromolecules, Experimental probes

Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers
Francesco Mosconi, Jean Franc¸ois Allemand, David Bensimon, and Vincent Croquette
We deduced the torque applied on a single stretched and twisted DNA by integrating the change in the molecule’s extension with respect to force as it is coiled. While consistent with previous direct measurements of the torque at high forces (F > 1 pN), this method, which is simple and does not require a sophisticated setup, allows for lower force estimates. We used this approach to deduce the effective torsional modulus of DNA, which decreases with force, and to estimate the buckling torque of DNA as a function of force in various salt conditions.
JPEGs: Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers.jpg (527.58 KB)
PDFs: Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers.pdf (8.96 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Experimental probes

Collaborative coupling between polymerase and helicase for leading-strand synthesis
Maria Manosas, Michelle M. Spiering, Fangyuan Ding, Vincent Croquette and Stephen J. Benkovic
Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.
JPEGs: Collaborative coupling between polymerase.jpg (1.06 MB)
PDFs: Collaborative coupling between polymerase.pdf (7.69 MB)
Research categories: Biological Macromolecules, Cellular mechanics, Experimental probes

Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination
Deepak Kumar Sinha, Pierre Neveu, Nathalie Gagey, Isabelle Aujard, Chouaha Benbrahim-Bouzidi, Thomas Le Saux, Christine Rampon, Carole Gauron, Bernard Goetz, Sylvie Dubruille, Marc Baaden, Michel Volovitch, David Bensimon, Sophie Vriz and Ludovic Jullien
We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ERT2). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ERT2 receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.
JPEGs: Photocontrol of Protein Activity in Cultured Cells.jpg (348.34 KB)
PDFs: Photocontrol of Protein Activity in Cultured Cells.pdf (6.06 MB)
Research categories: Tissues and Organisms, Experimental probes

One-dimensional deterministic transport in neurons measured by dispersion-relation phase spectroscopy
Ru Wang , Zhuo Wang, Joe Leigh, Nahil Sobh, Larry Millet, Martha U Gillette, Alex J Levine and Gabriel Popescu
Professor Levine, working with experimental colleagues at the University of Illinois at Urbana-Champaign, explores the “traffic jams” in transport of vesicles down the narrow neural filaments, axons and and dendrites.
JPEGs: One-dimensional deterministic transport.jpg (1.04 MB)
PDFs: One-dimensional-deterministic-transport.pdf (7.20 MB)
Research categories: Tissues and Organisms, Nonequilibrium physics, Experimental probes