Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria

BACKGROUND: Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases. METHODOLOGY: This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion. SIGNIFICANCE: Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria

Investigating the use of a hybrid plasmonic–photonic nanoresonator for optical trapping using finite-difference time-domain method

We investigate the use of a hybrid nanoresonator comprising a photonic crystal (PhC) cavity coupled to a plasmonic bowtie nanoantenna (BNA) for the optical trapping of nanoparticles in water. Using finite difference time-domain simulations, we show that this structure can confine light to an extremely small volume of ~30,000 nm3 (~30 zl) in the BNA gap whilst maintaining a high quality factor (5400–7700). The optical intensity inside the BNA gap is enhanced by a factor larger than 40 compared to when the BNA is not present above the PhC cavity. Such a device has potential applications in optical manipulation, creating high precision optical traps with an intensity gradient over a distance much smaller than the diffraction limit, potentially allowing objects to be confined to much smaller volumes and making it ideal for optical trapping of Rayleigh particles (particles much smaller than the wavelength of light)

High-pressure, high-temperature molecular doping of nanodiamond

The development of color centers in diamond as the basis for emerging quantum technologies has been limited by the need for ion implantation to create the appropriate defects. We present a versatile method to dope diamond without ion implantation by synthesis of a doped amorphous carbon precursor and transformation at high temperatures and high pressures. To explore this bottom-up method for color center generation, we rationally create silicon vacancy defects in nanodiamond and investigate them for optical pressure metrology. In addition, we show that this process can generate noble gas defects within diamond from the typically inactive argon pressure medium, which may explain the hysteresis effects observed in other high-pressure experiments and the presence of noble gases in some meteoritic nanodiamonds. Our results illustrate a general method to produce color centers in diamond and may enable the controlled generation of designer defects

Manipulating and assembling metallic beads with Optoelectronic Tweezers

Optoelectronic tweezers (OET) or light-patterned dielectrophoresis (DEP) has been developed as a micromanipulation technology for controlling micro- and nano-particles with applications such as cell sorting and studying cell communications. Additionally, the capability of moving small objects accurately and assembling them into arbitrary 2D patterns also makes OET an attractive technology for microfabrication applications. In this work, we demonstrated the use of OET to manipulate conductive silver-coated Poly(methyl methacrylate) (PMMA) microspheres (50 μm diameter) into tailored patterns. It was found that the microspheres could be moved at a max velocity of 3200 μm/s, corresponding to 4.2 nano-newton (10−9 N) DEP force, and also could be positioned with high accuracy via this DEP force. The underlying mechanism for this strong DEP force is shown by our simulations to be caused by a significant increase of the electric field close to the particles, due to the interaction between the field and the silver shells coating the microspheres. The associated increase in electrical gradient causes DEP forces that are much stronger than any previously reported for an OET device, which facilitates manipulation of the metallic microspheres efficiently without compromise in positioning accuracy and is important for applications on electronic component assembling and circuit construction