Project description:Manipulation of colloidal objects with light is important in diverse fields. While performance of traditional optical tweezers is restricted by the diffraction-limit, recent approaches based on plasmonic tweezers allow higher trapping efficiency at lower optical powers but suffer from the disadvantage that plasmonic nanostructures are fixed in space, which limits the speed and versatility of the trapping process. As we show here, plasmonic nanodisks fabricated over dielectric microrods provide a promising approach toward optical nanomanipulation: these hybrid structures can be maneuvered by conventional optical tweezers and simultaneously generate strongly confined optical near-fields in their vicinity, functioning as near-field traps themselves for colloids as small as 40?nm. The colloidal tweezers can be used to transport nanoscale cargo even in ionic solutions at optical intensities lower than the damage threshold of living micro-organisms, and in addition, allow parallel and independently controlled manipulation of different types of colloids, including fluorescent nanodiamonds and magnetic nanoparticles.
Project description:Optical tweezers offer a non-contact method for selecting single cells and translocating them from one microenvironment to another. We have characterized the optical tweezing of yeast S. cerevisiae and can manipulate single cells at 0.41 ± 0.06 mm/s using a 26.8 ± 0.1 mW from a 785 nm diode laser. We have fabricated and tested three cell isolation devices; a micropipette, a PDMS chip and a laser machined fused silica chip and we have isolated yeast, single bacteria and cyanobacteria cells. The most effective isolation was achieved in PDMS chips, where single yeast cells were grown and observed for 18 h without contamination. The duration of budding in S. cerevisiae was not affected by the laser parameters used, but the time from tweezing until the first budding event began increased with increasing laser energy (laser power × time). Yeast cells tweezed using 25.0 ± 0.1 mW for 1 min were viable after isolation. We have constructed a micro-consortium of yeast cells, and a co-culture of yeast and bacteria, using optical tweezers in combination with the PDMS network of channels and isolation chambers, which may impact on both industrial biotechnology and understanding pathogen dynamics.
Project description:Tethers were created between a living Escherichia coli bacterium and a bead by unspecifically attaching the bead to the outer membrane and pulling it away using optical tweezers. Upon release, the bead returned to the bacterium, thus showing the existence of an elastic tether between the bead and the bacterium. These tethers can be tens of microns long, several times the bacterial length. Using mutants expressing different parts of the outer membrane structure, we have shown that an intact core lipopolysaccharide is a necessary condition for tether formation, regardless of whether the beads were uncoated polystyrene or beads coated with lectin. A physical characterization of the tethers has been performed yielding visco-elastic tether force-extension relationships: for first pull tethers, a spring constant of 10-12 pN/mum describes the tether visco-elasticity, for subsequent pulls the spring constant decreases to 6-7 pN/mum, and typical relaxation timescales of hundreds of seconds are observed. Studies of tether stability in the presence of proteases, lipases, and amylases lead us to propose that the extracted tether is primarily composed of the asymmetric lipopolysaccharide containing bilayer of the outer membrane. This unspecific tethered attachment mechanism could be important in the initiation of bacterial adhesion.
Project description:Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.
Project description:Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.
Project description:The elastic properties of cell membranes, particularly the membrane tension and bending modulus, are known to be key regulators of cellular functions. Here, we present a correlative and integrated tool based on optical tweezers and scanning electron microscopy to accurately determine these properties in a variety of cell types. Although there are intrinsic difficulties associated with correlative experiments, we believe that the methods presented can be considered a suitable protocol for determining the elastic properties of cell membranes. For complete details on the use and execution of this protocol, please refer to Soares et al. (2020).
Project description:Over the past decade, optical tweezers (OT) have been increasingly used in neuroscience for studies of molecules and neuronal dynamics, as well as for the study of model organisms as a whole. Compared to other areas of biology, it has taken much longer for OT to become an established tool in neuroscience. This is, in part, due to the complexity of the brain and the inherent difficulties in trapping individual molecules or manipulating cells located deep within biological tissue. Recent advances in OT, as well as parallel developments in imaging and adaptive optics, have significantly extended the capabilities of OT. In this review, we describe how OT became an established tool in neuroscience and we elaborate on possible future directions for the field. Rather than covering all applications of OT to neurons or related proteins and molecules, we focus our discussions on studies that provide crucial information to neuroscience, such as neuron dynamics, growth, and communication, as these studies have revealed meaningful information and provide direction for the field into the future.
Project description:Dynamic arrays of microbeads and cells offer great flexibility and potential as platforms for sensing and manipulation applications in various scientific fields, especially biology and medicine. Here, we present a simple method for assembling and manipulating dense dynamic arrays based on time-shared scanning optical tweezers with a microlens array. Three typical examples, including the dynamic and simultaneous bonding of microbeads in real-time, are demonstrated. The optical design and the hardware setup for our approach are also described.
Project description:Class V myosin (myosin-V) is a cargo transporter that moves along an actin filament with large (approximately 36-nm) successive steps. It consists of two heads that each includes a motor domain and a long (23 nm) neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics) of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 k(B)T from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.
Project description:Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.