Analysis of the precision, robustness and speed of elastic resonator interference stress microscopy

This project has received funding from the Human Frontiers Science Program (RGY0074/2013), the Scottish Funding Council (via SUPA), the EPSRC DTP (EP/L505079/1), a BBSRC research grant (BB/P027148/1), an EPSRC programme grant (EP/P030017/1) and the RS MacDonald Charitable Trust.Localization-microscopy-based methods are widely used to map the forces that cells apply to their substrates and to study important questions of cellular biomechanics. By contrast, elastic resonator interference stress microscopy (ERISM) uses an interference-based approach, which requires low light intensity and facilitates imaging of cellular forces with extreme precision (down to pN forces) and robustness (e.g., for continuous force monitoring over weeks). Here, the measurement trade-offs and numerical considerations required to optimize the performance of ERISM are described. The crucial parts of the fitting algorithm and the computational tools used to evaluate the data are explained in detail, and the precision and accuracy achievable with ERISM are analyzed. Additional features that can improve the robustness of ERISM further are discussed. The implementation of the analysis algorithm is verified with simulated test data and with experimental data. In addition, an approach to increase the acquisition speed of ERISM by a factor of four compared to the original implementation is described. In combination, these strategies allow us to measure the forces generated by a neural growth cone with high temporal resolution and continuously over several hours.PostprintPeer reviewe

KIAA0319 influences cilia length, cell migration and mechanical cell-substrate interaction

Funding: This work was supported by Action Medical Research/ The Chief Scientist (CSO) Office, Scotland [GN 2614], Royal Society [RG160373], Carnegie Trust [50341], Wellcome Trust ISSF grant 105621/Z/14/Z, and RS Macdonald Charitable Trust grants to SP and Engineering and Physical Sciences Research Council [EP/P030017/1], Biotechnology and Biological Sciences Research Council [BB/P027148/1], and the European Research Council Starting Grant ABLASE [640012] grants to MCG. SP is a Royal Society University Research Fellow.Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell–substrate force regulation.Publisher PDFPeer reviewe

An automated framework of inner segment/outer segment defect detection for retinal SD-OCT images

The integrity of inner segment/outer segment (IS/OS) has high correlation with lower visual acuity in patients suffering from blunt trauma. An automated 3D IS/OS defect detection method based on the SD-OCT images was proposed. First, 11 surfaces were automatically segmented using the multiscale 3D graph-search approach. Second, the sub-volumes between surface 7 and 8 containing IS/OS region around the fovea (diameter of mm) were extracted and flattened based on the segmented retinal pigment epithelium layer. Third, 5 kinds of texture based features were extracted for each voxel. A KNN classifier was trained and each voxel was classified as disrupted or nondisrupted and the responding defect volume was calculated. The proposed method was trained and tested on 9 eyes from 9 trauma subjects using the leave-one-out cross validation method. The preliminary results demonstrated the feasibility and efficiency of the proposed method

Lasing within live cells containing intracellular optical microresonators for barcode-type cell tagging and tracking

This work was supported by the European Union Marie Curie Career Integration Grant (PCIG12-GA-2012-334407) and the Scottish Funding Council (SUPA II). M.S. acknowledges funding by the German Science Foundation (DFG) through a Research Fellowship (SCHU 3003/1-1).We report on a laser that is fully embedded into a single live cell. By harnessing natural endocytosis of the cell we introduce a fluorescent whispering gallery mode (WGM) micro-resonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells. Our approach can be applied to different cell types and cells with micro-resonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking of individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing and adaptive imaging.PostprintPeer reviewe

Foreword

Funding: European Research Council under the European Union’s Horizon 2020 Framework Programme (ERC StG ABLASE, 640012); BBSRC (BB/P027148/1); EPSRC Programme Grant (EP/P030017/1); EPSRC Doctoral Training Partnership (EP/N509759/1, EP/L505079/1).Mechanobiology plays a prominent role in cancer invasion and metastasis. The ability of a cancer to degrade extracellular matrix (ECM) is likely connected to its invasiveness. Many cancer cells form invadopodia—micrometer-sized cellular protrusions that promote invasion through matrix degradation (proteolysis). Although it has been hypothesized that invadopodia exert mechanical force that is implicated in cancer invasion, direct measurements remain elusive. Here, we use a recently developed interferometric force imaging technique that provides piconewton resolution to quantify invadopodial forces in cells of head and neck squamous carcinoma and to monitor their temporal dynamics. We compare the force exerted by individual protrusions to their ability to degrade ECM and investigate the mechanical effects of inhibiting invadopodia through overexpression of microRNA-375. By connecting the biophysical and biochemical characteristics of invadopodia, our study provides a new perspective on cancer invasion that, in the future, may help to identify biomechanical targets for cancer therapy.Publisher PDFPeer reviewe

Podocyte injury elicits loss and recovery of cellular forces

K.E. Haley was funded by a University of St. Andrews 600th Anniversary PhD Scholarship. M.C.G. acknowledges support by the Human Frontiers Science Program (RGY0074/2013), the Scottish Funding Council (via SUPA), EPSRC (EP/P030017/1) and the ERC Starting Grant ABLASE (640012). D.J.H. was supported by NHS Lothian. M.C.G. and P.A.R. acknowledge support by the BBSRC (BB/P027148/1).In the healthy kidney, specialized cells called podocytes form a sophisticated blood filtration apparatus that allows excretion of wastes and excess fluid from the blood while preventing loss of proteins such as albumin. To operate effectively, this filter is under substantial hydrostatic mechanical pressure. Given their function, it is expected that the ability to apply mechanical force is crucial to the survival of podocytes. However, to date podocyte mechanobiology remains poorly understood, largely due to a lack of experimental data on the forces involved. Herein, we perform quantitative, continuous, non-disruptive and high-resolution measurement of the forces exerted by differentiated podocytes in real time using a recently introduced functional imaging modality for continuous force mapping. Using an accepted model for podocyte injury, we find that injured podocytes experience near complete loss of cellular force transmission, but that this is reversible under certain conditions. The observed changes in force correlate with F-actin rearrangement and reduced expression of podocyte-specific proteins. By introducing robust and high-throughput mechanical phenotyping and by demonstrating the significance of mechanical forces in podocyte injury, this research paves the way to a new level of understanding of the kidney. In addition, we integrate cellular force measurements with immunofluorescence and perform continuous long-term force measurements of a cell population, which has not been feasible with established force mapping techniques. As such, our approach has general applicability to a wide range of biomedical questions involving mechanical forces.Publisher PDFPeer reviewe

Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy

This project has received funding from the Human Frontiers Science Program (RGY0074/2013), the Scottish Funding Council (via SUPA), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 640012), the EPSRC DTP (EP/L505079/1), the RS MacDonald Charitable Trust and the MRC (G1100116 and G110312/1).Cellular forces are crucial for many biological processes but current methods to image them have limitations with respect to data analysis, resolution and throughput. Here, we present a robust approach to measure mechanical cell–substrate interactions in diverse biological systems by interferometrically detecting deformations of an elastic micro-cavity. Elastic resonator interference stress microscopy (ERISM) yields stress maps with exceptional precision and large dynamic range (2 nm displacement resolution over a >1 μm range, translating into 1 pN force sensitivity). This enables investigation of minute vertical stresses (<1 Pa) involved in podosome protrusion, protein-specific cell–substrate interaction and amoeboid migration through spatial confinement in real time. ERISM requires no zero-force reference and avoids phototoxic effects, which facilitates force monitoring over multiple days and at high frame rates and eliminates the need to detach cells after measurements. This allows observation of slow processes such as differentiation and further investigation of cells, for example, by immunostaining.PostprintPeer reviewe

Influence of cavity thickness and emitter orientation on the efficiency roll-off of phosphorescent organic light-emitting diodes

This article describes the first systematic investigation of how the efficiency roll-off in organic light-emitting diodes (OLEDs) is influenced by the position and orientation of the emitter molecules within the OLED cavity. The efficiency roll-off is investigated for two OLED stacks containing either the phosphorescent emitter Ir(MDQ)2(acac) or Ir(ppy)3 by varying the distance between emitter and metal cathode; a strong influence of emitter position and orientation on roll-off is observed. The measurements are modeled by triplet-triplet-annihilation (TTA) theory yielding the critical current density and the TTA rate constant. It is found that Ir(MDQ)2(acac) shows the lowest roll-off when the emitter is located in the first optical maximum of the electromagnetic field, whereas the roll-off of the Ir(ppy)3 stack is lowest when the emitter is positioned closer to the metal cathode. Measurement and modeling of time-resolved electroluminescence show that the different roll-off behavior is due to the different orientation and the corresponding change of the decay rate of the emissive dipoles of Ir(MDQ)2(acac) and Ir(ppy)3. Finally, design principles are developed for optimal high-brightness performance by modeling the roll-off as a function of emitter-cathode distance, emissive dipole orientation, and radiative efficiency.PostprintPeer reviewe

Elastic-resonator-interference-stress-microscopy (ERISM)

The forces biological cells apply to their environment are recognised to be critical during processes like migration, division, wound healing, and stem cell differentiation. Methods to measure these forces have been extremely valuable in contributing to our understanding of cell-substrate and cell-cell interactions. However, existing force sensing techniques struggle to measure forces cells apply perpendicular to the plane of their substrate although these out-of-plane forces have been demonstrated to be important in many processes. In addition, most currently used force sensing techniques require fluorescence imaging which can lead to photo-toxic effects if high frame rates are required. Finally, many methods require detaching of cells after the measurement which prevents measuring the same cells repeatedly or using immunostaining, which is an important tool for linking biomechanical and biochemical observations. In this thesis, we introduce a novel high-throughput and low-light-intensity force sensing technique which is inherently well suited to measure vertical forces. Elastic-Resonator-Interference-Stress-Microscopy (ERISM) measures the spatially resolved reflectance of an elastic micro-cavity. With fully automated hyperspectral imaging and data analysis supported by transfer-matrix modelling, this allows tracking of nanometre thickness changes across a large area of the cavity. By combining Atomic-Force-Microscopy with a Finite-Element-Method, we extract basic material properties of the micro-cavities to calculate stress and force. Using the example of different neural cells, we provide experimental evidence that ERISM measurements can be performed over hours at high frame rates or repeatedly over weeks for the same sample to investigate a variety of cellular processes like cell spreading, growth cone migration, and stem cell differentiation. We perform immunostaining for cell specific markers on ERISM micro-cavities as detaching of the cells is not required. Furthermore, we find that the high throughput of ERISM allows us to find significant differences between wild-type and knock-out cell populations for a gene associated with dyslexia