High resolution photonic force microscopy based on sharp nano-fabricated tips

Sub-nm resolution images can be achieved by Atomic Force Microscopy (AFM) on samples that are deposited on hard substrates. However, it is still extremely challenging to image soft interfaces, such as biological membranes, due to the deformations induced by the tip. Photonic Force Microscopy (PhFM), based on optical tweezers (OT), represents an interesting alternative for soft scanning-probe microscopy. Using light instead of a physical cantilever to hold the scanning probe results in a stiffness ($k_{OT}\sim0.1-0.001$ pN/nm) which can be 2-3 orders of magnitude lower than that of standard cantilevers ($k_{AFM}\sim 10$ pN/nm). Combined with nm resolution of displacement measurements of the trapped probe, this allows for imaging soft materials without force-induced artefacts. However, the size of the optically trapped probe, often chosen as a $\sim \mu$m-size sphere, has so far limited the resolution of PhFM. Here we show a novel and simple nanofabrication protocol to massively produce optically trappable quartz particles which mimic the sharp tips of AFM. We demonstrate and quantify the stable trapping of particles with tips as sharp as 35 nm, the smallest used in PhFM to date. Raster scan images of rigid nanostructures with features smaller than 80 nm obtained with our tips compare well with AFM images of the same samples. Imaging the membrane of living malaria-infected red blood cells produces no visible artefacts and reveals the sub-micron structural features termed knobs, related to the parasite activity within the cell. The use of nano-engineered particles in PhFM opens the way to imaging soft and biological samples at high resolution

Signature of (anti)cooperativity in the stochastic fluctuations of small systems: application to the bacterial flagellar motor

The cooperative binding of molecular agents onto a substrate is pervasive in living systems. To study whether a system shows cooperativity, one can rely on a fluctuation analysis of quantities such as the number of substrate-bound units and the residence time in an occupancy state. Since the relative standard deviation from the statistical mean monotonically decreases with the number of binding sites, these techniques are only suitable for small enough systems, such as those implicated in stochastic processes inside cells. Here, we present a general-purpose grand canonical Hamiltonian description of a small one-dimensional (1D) lattice gas with either nearest-neighbor or long-range interactions as prototypical examples of cooperativity-influenced adsorption processes. First, we elucidate how the strength and sign of the interaction potential between neighboring bound particles on the lattice determine the intensity of the fluctuations of the mean occupancy. We then employ this relationship to compare the theoretical predictions of our model to data from single molecule experiments on bacterial flagellar motors (BFM) of E. coli. In this way, we find evidence that cooperativity controls the mechano-sensitive dynamical assembly of the torque-generating units, the so-called stator units, onto the BFM. Furthermore, in an attempt to quantify fluctuations and the adaptability of the BFM, we estimate the stator-stator interaction potential. Finally, we conclude that the system resides in a sweet spot of the parameter space (phase diagram) suitable for a smoothly adaptive system while minimizing fluctuations.Comment: 35 pages, 18 figures, 4 table

Phenotyping male infertility in the mouse: how to get the most out of a ‘non-performer’

BACKGROUND: Functional male gametes are produced through complex processes that take place within the testis, epididymis and female reproductive tract. A breakdown at any of these phases can result in male infertility. The production of mutant mouse models often yields an unexpected male infertility phenotype. It is with this in mind that the current review has been written. The review aims to act as a guide to the 'non-reproductive biologist' to facilitate a systematic analysis of sterile or subfertile mice and to assist in extracting the maximum amount of information from each model. METHODS: This is a review of the original literature on defects in the processes that take a mouse spermatogonial stem cell through to a fully functional spermatozoon, which result in male infertility. Based on literature searches and personal experience, we have outlined a step-by-step strategy for the analysis of an infertile male mouse line. RESULTS: A wide range of methods can be used to define the phenotype of an infertile male mouse. These methods range from histological methods such as electron microscopy and immunohistochemistry, to hormone analyses and methods to assess sperm maturation status and functional competence. CONCLUSION: With the increased rate of genetically modified mouse production, the generation of mouse models with unexpected male infertility is increasing. This manuscript will help to ensure that the maximum amount of information is obtained from each mouse model and, by extension, will facilitate the knowledge of both normal fertility processes and the causes of human infertility

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

A simple low-cost device enables four epi-illumination techniques on standard light microscopes

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy

Dynamic stiffening of the flagellar hook

Many bacteria are motile by means of one or more rotating rigid helical flagella, making them the only known organism to use rotation as a means of propulsion. The rotation is supplied by the bacterial flagellar motor, a particularly powerful rotary molecular machine. At the base of each flagellum is the hook, a soft helical polymer that acts as a universal joint, coupling rotation of the rigid membrane-spanning rotor to rotation of the rigid extra-cellular flagellum. In multi-flagellated bacterial species, where thrust is provided by a hydrodynamically coordinated bundle of flagella, the flexibility of the hook is particularly crucial, as many of the flagella within the bundle rotate significantly off-axis from their motor. But, consequently, the thrust produced by a single rotating flagellum applies a significant bending moment to the hook. So, the hook needs to simultaneously provide the compliance necessary for off-axis bundle formation and the rigidity necessary to withstand the large hydrodynamical forces of swimming. To elucidate how the hook can fulfill this double functionality, measurements of the mechanical behavior of individual hooks under dynamical conditions are needed. Here, via new high-resolution measurements and a novel analysis of hook fluctuations during in vivo motor rotation in bead assays, we resolve the elastic response of single hooks under increasing torsional stress, revealing a clear dynamic increase in their bending stiffness. Accordingly, the persistence length of the hook increases by more than one order of magnitude with applied torque. Such strain-stiffening allows the system to be flexible when needed yet reduce deformation under high loads, allowing cellular motility at high speed