Force measurements with optical tweezers inside living cells

The force exerted by optical tweezers can be measured by tracking the momentum changes of the trapping beam, a method which is more general and powerful than traditional calibration techniques as it is based on first principles, but which has not been brought to its full potential yet, probably due to practical difficulties when combined with high-NA optical traps, such as the necessity to capture a large fraction of the scattered light. We show that it is possible to measure forces on arbitrary biological objects inside cells without an in situ calibration, using this approach. The instrument can be calibrated by measuring three scaling parameters that are exclusively determined by the design of the system, thus obtaining a conversion factor from volts to piconewtons that is theoretically independent of the physical properties of the sample and its environment. We prove that this factor keeps valid inside cells as it shows good agreement with other calibration methods developed in recent years for viscoelastic media. Finally, we apply the method to measuring the stall forces of kinesin and dynein in living A549 cells.Publisher PD

HoloTrap: Interactive hologram design for multiple dynamic optical trapping

This work presents an application that generates real-time holograms to be displayed on a holographic optical tweezers setup; a technique that allows the manipulation of particles in the range from micrometres to nanometres. The software is written in Java, and uses random binary masks to generate the holograms. It allows customization of several parameters that are dependent on the experimental setup, such as the specific characteristics of the device displaying the hologram, or the presence of aberrations. We evaluate the software's performance and conclude that real-time interaction is achieved. We give our experimental results from manipulating 5 micron-diametre microspheres using the program.Comment: 17 pages, 6 figure

Design strategies for optimizing holographic optical tweezers setups

We provide a detailed account of the construction of a system of holographic optical tweezers. While much information is available on the design, alignment and calibration of other optical trapping configurations, those based on holography are relatively poorly described. Inclusion of a spatial light modulator in the setup gives rise to particular design trade-offs and constraints, and the system benefits from specific optimization strategies, which we discuss.Comment: 16 pages, 15 figure

Evidence of telomere attrition and a potential role for DNA damage in systemic sclerosis

[Background]: To investigate the role of cell senescence in systemic sclerosis (SSc), we analyzed telomere shortening (TS) in SSc patients and the effect of targeting DNA damage in the bleomycin model of skin fibrosis. [Results]: Telomere length (TL) in blood leukocytes of 174 SSc patients and 68 healthy controls was measured by Southern blot, and we found shorter age-standardized TL in SSc patients compared to healthy controls. TL was shorter in SSc patients with ILD compared to those without ILD and in anti-topoisomerase I positive compared to anti-centromere positive patients. To analyze the potential role of DNA damage in skin fibrosis, we evaluated the effects of the DNA protective GSE4 peptide in the bleomycin mouse model of scleroderma and the fibrotic response of cultured human dermal fibroblasts. Administration of GSE4-nanoparticles attenuated bleomycin-induced skin fibrosis as measured by Masson’s staining of collagen and reduced Acta2 and Ctgf mRNA expression, whereas transduction of dermal fibroblasts with a lentiviral GSE4 expression vector reduced COL1A1, ACTA2 and CTGF gene expression after stimulation with bleomycin or TGF-β, in parallel to a reduction of the phospho-histone H2A.X marker of DNA damage. [Conclusions]: SSc is associated with TS, particularly in patients with lung disease or anti-topoisomerase I antibodies. Administration of GSE4 peptide attenuated experimental skin fibrosis and reduced fibroblast expression of profibrotic factors, supporting a role for oxidative DNA damage in scleroderma.The authors received financial support from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (PI19/01129, PI20/00335, and RIER network RD16/0012 RETICS program), co-financed by the European Regional Development Fund (FEDER)

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density.

OBJECTIVES: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. METHODS: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. RESULTS: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10-9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. CONCLUSION: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism

Atomic Species Associated with the Portevin–Le Chatelier Effect in Superalloy 718 Studied by Mechanical Spectroscopy

In many Ni-based superalloys, dynamic strain aging (DSA) generates an inhomogeneous plastic deformation resulting in jerky flow known as the Portevin--Le Chatelier (PLC) effect. This phenomenon has a deleterious effect on the mechanical properties and, at high temperature, is related to the diffusion of substitutional solute atoms toward the core of dislocations. However, the question about the nature of the atomic species responsible for the PLC effect at high temperature still remains open. The goal of the present work is to answer this important question; to this purpose, three different 718-type and a 625 superalloy were studied through a nonconventional approach by mechanical spectroscopy. The internal friction (IF) spectra of all the studied alloys show a relaxation peak P718 (at 885 K for 0.1 Hz) in the same temperature range, 700 K to 950 K, as the observed PLC effect. The activation parameters of this relaxation peak have been measured, Ea(P718){\thinspace}={\thinspace}2.68{\thinspace}{\textpm}{\thinspace}0.05 eV, $\tau$0{\thinspace}={\thinspace}2{\textperiodcentered}10-15 {\textpm} 1 s as well as its broadening factor $\beta${\thinspace}={\thinspace}1.1. Experiments on different alloys and the dependence of the relaxation strength on the amount of Mo attribute this relaxation to the stress-induced reorientation of Mo-Mo dipoles due to the short distance diffusion of one Mo atom by exchange with a vacancy. Then, it is concluded that Mo is the atomic species responsible for the high-temperature PLC effect in 718 superallo