Direct observation of mammalian cell growth and size regulation

We introduce a microfluidic system for simultaneously measuring single cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 hours of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1/S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis

A Mechanical Mass Sensor with Yoctogram Resolution

Nanoelectromechanical systems (NEMS) have generated considerable interest as inertial mass sensors. NEMS resonators have been used to weigh cells, biomolecules, and gas molecules, creating many new possibilities for biological and chemical analysis [1-4]. Recently, NEMS-based mass sensors have been employed as a new tool in surface science in order to study e.g. the phase transitions or the diffusion of adsorbed atoms on nanoscale objects [5-7]. A key point in all these experiments is the ability to resolve small masses. Here we report on mass sensing experiments with a resolution of 1.7 yg (1 yg = 10^-24 g), which corresponds to the mass of one proton, or one hydrogen atom. The resonator is made of a ~150 nm long carbon nanotube resonator vibrating at nearly 2 GHz. The unprecedented level of sensitivity allows us to detect adsorption events of naphthalene molecules (C10H8) and to measure the binding energy of a Xe atom on the nanotube surface (131 meV). These ultrasensitive nanotube resonators offer new opportunities for mass spectrometry, magnetometry, and adsorption experiments.Comment: submitted version of the manuscrip

Comprehensive characterization of molecular interactions based on nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions

Cavity optomechanics on a microfluidic resonator with water and viscous liquids

Currently, optical- or mechanical-resonances are commonly used in microfluidic research. However, optomechanical oscillations by light pressure were not shown with liquids. This is because replacing the surrounding air with water inherently increases the acoustical impedance and hence the associated acoustical radiation-losses. Here, we bridge between microfluidics and optomechanics by fabricating hollow bubble resonators with liquid inside and optically exciting 100-MHz vibrations with only mW optical-input power. This constitutes the first time that any microfluidic system is optomechanically actuated. We further prove the feasibility of microfluidic optomechanics on liquids by demonstrating vibrations on organic fluids with viscous-dissipation higher than blood viscosity while measuring density changes in the liquid via the vibration frequency shift. Our device will enable using cavity optomechanics for studying non-solid phases of matter

Detecting single viruses and nanoparticles using whispering gallery microlasers

Detection and characterization of individual nano-scale particles, virions, and pathogens are of paramount importance to human health, homeland security, diagnostic and environmental monitoring[1]. There is a strong demand for high-resolution, portable, and cost-effective systems to make label-free detection and measurement of individual nanoparticles, molecules, and viruses [2-6]. Here, we report an easily accessible, real-time and label-free detection method with single nanoparticle resolution that surpasses detection limit of existing micro- and nano-photonic devices. This is achieved by using an ultra-narrow linewidth whispering gallery microlaser, whose lasing line undergoes frequency splitting upon the binding of individual nano-objects. We demonstrate detection of polystyrene and gold nanoparticles as small as 15 nm and 10 nm in radius, respectively, and Influenza A virions by monitoring changes in self-heterodyning beat note of the split lasing modes. Experiments are performed in both air and aqueous environment. The built-in self-heterodyne interferometric method achieved in a microlaser provides a self-reference scheme with extraordinary sensitivity [7,8], and paves the way for detection and spectroscopy of nano-scale objects using micro- and nano-lasers.Comment: Main Text: 14 pages, 5 figures, 27 references. Supplement: 26 pages, 12 figures, 26 reference

On-chip Single Nanoparticle Detection and Sizing by Mode Splitting in an Ultra-high-Q Microresonator

The ability to detect and size individual nanoparticles with high resolution is crucial to understanding behaviours of single particles and effectively using their strong size-dependent properties to develop innovative products. We report real-time, in-situ detection and sizing of single nanoparticles, down to 30 nm in radius, using mode-splitting in a monolithic ultra-high-Q whispering-gallery-mode (WGM) microtoroid resonator. Particle binding splits a WGM into two spectrally shifted resonance modes, forming a self-referenced detection scheme. This technique provides superior noise suppression and enables extracting accurate size information in a single-shot measurement. Our method requires neither labelling of the particles nor apriori information on their presence in the medium, providing an effective platform to study nanoparticles at single particle resolution.Comment: 23 pages, 8 figure

Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans

Spontaneous mechanical oscillation of a DC driven single crystal

There is a large interest to decrease the size of mechanical oscillators since this can lead to miniaturization of timing and frequency referencing devices, but also because of the potential of small mechanical oscillators as extremely sensitive sensors. Here we show that a single crystal silicon resonator structure spontaneously starts to oscillate when driven by a constant direct current (DC). The mechanical oscillation is sustained by an electrothermomechanical feedback effect in a nanobeam, which operates as a mechanical displacement amplifier. The displacement of the resonator mass is amplified, because it modulates the resistive heating power in the nanobeam via the piezoresistive effect, which results in a temperature variation that causes a thermal expansion feedback-force from the nanobeam on the resonator mass. This self-amplification effect can occur in almost any conducting material, but is particularly effective when the current density and mechanical stress are concentrated in beams of nano-scale dimensions

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depo-lymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites