Interference Reflection Microscopy Research Articles

Multicamera simultaneous total internal reflection and interference reflection microscopy.

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multicolour total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilises a camera for each channel (IRM and TIRF) allowing independent optimisation of camera parameters for the two different modalities. We illustrate its application by imaging unlabelled microtubules and GFP-labelled end-binding protein EB1, which forms comets on the tips of polymerising microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.

Collective effect of Vigna sp. (mung) tubulin GTP hydrolysis rate divergence on microtubule filament assembly.

Microtubules (MTs) are dynamic cytoskeletal filaments with highly conserved sequences across evolution, polymerizing by the GTP-dependent assembly of tubulin subunits. Despite the sequence conservation, MT polymerization kinetics diverge quantitatively between vertebrate brain, the model plant Arabidopsis and the protozoan Plasmodium. Previously, tubulin purified from seedlings of the plant Vigna sp. (mung) by temperature cycling was found to have a very low critical concentration. However, the lengths of MTs were sub-micron, much shorter than brain tubulin filaments. This was explained in simulations to be the result of the collective effect of high nucleation and GTP hydrolysis rates. Here, we test the effect of GTPase rates of affinity-purified Vigna sp. tubulin on microtubule polymerization and elongation. Affinity-purified mung tubulin is active and has a critical concentration of .37 μM. The GTP-dependent polymerization kinetics are transient, consistent with previous results. Polymerization is stabilized in the presence of either GTP analog GMPPNP (non-hydrolyzable) or GMPCPP (slow-hydrolyzable). Using interference reflection microscopy (IRM) we find polymerization with the non-hydrolysable analog significantly increases filament numbers, while lengths are unaffected for both GTP analogs. However, prolonged incubation with slow-hydrolyzable GMPCPP results in long filaments, pointing to GTP hydrolysis as a key factor determining MT length. We find the average GTPase turnover number of mung tubulin is 22.8 min-1, compared to 2.04 min-1 for goat brain tubulin. Thus modulating GTPase rates affects both nucleation and elongation. This quantitative divergence in kinetics despite high sequence conservation in the GTPase domains of α- and β-tubulin could help better understand the roles of selective pressure and function in the diverse organisms.

Multi-camera Simultaneous Total Internal Reflection and Interference Reflection Microscopy.

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25 nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multi-color total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilizes a camera for each channel (IRM and TIRF) allowing independent optimization of camera parameters for the two different modalities. We illustrate its application by imaging unlabeled microtubules and GFP-labeled end-binding protein EB1 which forms comets on the tips of polymerizing microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.

High-Throughput Combinatorial Analysis of the Spatiotemporal Dynamics of Nanoscale Lithium Metal Plating.

The development of Li metal batteries requires a detailed understanding of complex nucleation and growth processes during electrodeposition. In situ techniques offer a framework to study these phenomena by visualizing structural dynamics that can inform the design of uniform plating morphologies. Herein, we combine scanning electrochemical cell microscopy (SECCM) with in situ interference reflection microscopy (IRM) for a comprehensive investigation of Li nucleation and growth on lithiophilic thin-film gold electrodes. This multimicroscopy approach enables nanoscale spatiotemporal monitoring of Li plating and stripping, along with high-throughput capabilities for screening experimental conditions. We reveal the accumulation of inactive Li nanoparticles in specific electrode regions, yet these regions remain functional in subsequent plating cycles, suggesting that growth does not preferentially occur from particle tips. Optical-electrochemical correlations enabled nanoscale mapping of Coulombic Efficiency (CE), showing that regions prone to inactive Li accumulation require more cycles to achieve higher CE. We demonstrate that electrochemical nucleation time (tnuc) is a lagging indicator of nucleation and introduce an optical method to determine tnuc at earlier stages with nanoscale resolution. Plating at higher current densities yielded smaller Li nanoparticles and increased areal density, and was not affected by heterogeneous topographical features, being potentially beneficial to achieve a more uniform plating at longer time scales. These results enhance the understanding of Li plating on lithiophilic surfaces and offer promising strategies for uniform nucleation and growth. Our multimicroscopy approach has broad applicability to study nanoscale metal plating and stripping phenomena, with relevance in the battery and electroplating fields.

Estimate of entropy production rate can spatiotemporally resolve the active nature of cell flickering

We use the short-time inference scheme [Manikandan , ], obtained within the framework of stochastic thermodynamics, to infer a lower bound to entropy production rate from flickering data generated by interference reflection microscopy of HeLa cells. We can clearly distinguish active cell membranes from their adenosine-triphosphate-depleted selves and even spatiotemporally resolve activity down to the scale of about 1 µm. Our estimate of activity is . Published by the American Physical Society 2024

Printing, Characterizing, and Assessing Transparent 3D Printed Lenses for Optical Imaging

AbstractHigh‐quality lens production has involved subtractive manufacturing methods for centuries. These methods demand specialist equipment and expertise that often render custom high‐grade glass optics inaccessible. A low‐cost, accessible, and reproducible method is developed to manufacture high‐quality three dimensional (3D) printed lenses using consumer‐grade technology. Various planoconvex lenses are produced using a consumer‐grade 3D printer and low‐cost spin coating setup, and printed lenses are compared to commercial glass counterparts. A range of mechanical and optical methods are introduced to determine the surface quality and curvature of 3D printed lenses. Amongst others, high‐resolution interference reflection microscopy methods are used to reconstruct the convex surface of printed lenses and quantify their radius of curvature. The optical throughput and performance of 3D printed lenses are assessed using optical transmissivity measurements and classical beam characterization methods. It is determined that 3D printed lenses have comparable curvature and performance to commercial glass lenses. Finally, the application of 3D printed lenses is demonstrated for brightfield transmission microscopy, resolving sub‐cellular structures over a 2.3 mm field‐of‐view. The high reproducibility and comparable performance of 3D printed lenses present great opportunities for additive manufacturing of bespoke optics for low‐cost rapid prototyping and improved accessibility to high‐quality optics in low‐resource settings.

In Operando Study of Charge Modulation in MoS2 Transistors by Excitonic Reflection Microscopy.

Monolayers of transition metal dichalcogenides (2D TMDs) experience strong modulation of their optical properties when the charge density is varied. Indeed, the transition from carriers composed mostly of excitons at low electron density to a situation in which trions dominate at high density is accompanied by a significant evolution of both the refractive index and the extinction coefficient. Using optical interference reflection microscopy at the excitonic wavelength, this (n, κ)-q relationship can be exploited to directly image the electron density in operating TMD devices. In this work, we show how this technique, which we call XRM (excitonic reflection microscopy), can be used to study charge distribution in MoS2 field-effect transistors with subsecond throughput, in wide-field mode. Complete maps of the charge distribution in the transistor channel at any drain and gate bias polarization point (VDS, VGS) are obtained, at ∼3 orders of magnitude faster than with scanning probe techniques such as KPFM. We notably show how the advantages of XRM enable real-time mapping of bias-dependent charge inhomogeneities, the study of resistive delays in 2D polycrystalline networks, and the evaluation of the VDS vs VGS competition to control the charge distribution in active devices.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Mechano-regulation by clathrin pit-formation and passive cholesterol-dependent tubules during de-adhesion

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Polymer-induced adhesion of endothelial cells

The effects of neutral dextran concentration and molecular mass on the adhesion of endothelial cells (EC) to siliclad-covered glass surfaces were studied using interference reflection microscopy (IRM). Results indicate that close contact of the EC to the glass slides is markedly enhanced in the presence of 500 kDa dextran, with this increase reflected by both the speed of forming close contact as well as the size of the contact area. This increased adhesion is attributed to the reduction in surface concentrations of large polymers and, therefore, to the attractive forces caused by depletion interaction. Our findings suggest that depletion could play an important role in cell-cell or cell-surface interactions via accelerating and enhancing close contacts. This interaction should thus be considered in vivo and in vitro for specific potential applications, such as cell culture and cell adhesion to biomimetic surfaces. It should therefore be of particular interest in a wide range of biomedical applications.

Investigating the liquid Characterising the surface quality of 3D printed optical elements using interference reflection microscopy

Optical microscopy has used the same manufacturing methods for milling and sanding glass elements to produce high-quality optics for over a century. Recent developments in 3D printing have opened the possibility of printing optical components, but the surface quality of these 3D prints remains unknown.

A simple image processing pipeline to sharpen topology maps in multi-wavelength interference microscopy.

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps, and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed-cell specimens.

Optical Super‐Localisation of Single Nanoparticle Nucleation and Growth in Nanodroplets

AbstractThe formation of metal nanoparticles (NPs) on surfaces by electrodeposition is of significant interest, particularly with a view to understand the early stages of nucleation and growth. Here, the combination of scanning electrochemical cell microscopy (SECCM) and interference reflection microscopy (IRM) is demonstrated to be a compelling approach for real‐time monitoring of NP dynamics within the SECCM meniscus‐electrode wetted area, through synchronous monitoring in the millisecond range of the electrochemical and optical signatures. Diffraction‐limited entities, undergoing phase changes at the electrode substrate, are readily highlighted and tracked in time, including the onset time for the appearance of NPs and their movement over time. The results strongly implicate the rapid formation, surface diffusion and aggregation of smaller entities (not detectable optically) to produce the larger electrodeposited NPs. By applying SECCM tips of different size, it is also possible to understand how the wetted area (meniscus size) plays a key role in the number of NPs formed, with small tip sizes allowing the formation of single NPs. The SECCM‐IRM approach is expected to be a powerful platform for the study of myriad phase‐formation processes at the nanoscale, particularly by drawing on the possibility of making hundreds or thousands of measurements in fresh surface locations through SECCM technology.

Imaging solid-electrolyte interphase dynamics using operando reflection interference microscopy.

The quality of the solid-electrolyte interphase is crucial for the performance of most battery chemistries, but its formation dynamics during operation are not well understood due to a lack of reliable operando characterization techniques. Herein, we report a dynamic, non-invasive, operando reflection interference microscope to enable the real-time imaging of the solid-electrolyte interphase during its formation and evolution processes with high sensitivity. The stratified structure of the solid-electrolyte interphase formed during four distinct steps includes the emergence of a permanent inner inorganic layer enriched in LiF, a transient assembly of an interfacial electrified double layer and a consequent emergence of a temporary outer organic-rich layer whose presence is reversible with electrochemical cycling. Reflection interference microscope imaging reveals an inverse correlation between the thicknesses of two interphasial subcomponents, implying that the permanent inorganic-rich inner layer dictates the organic-rich outer layer formation and lithium nucleation. The real-time visualization of solid-electrolyte interphase dynamics provides a powerful tool for the rational design of battery interphases.

Dynamics of prolate spheroids in the vicinity of an air-water interface.

In this article, we present the mobilities of prolate ellipsoidal micrometric particles close to an air-water interface measured by dual wave reflection interference microscopy. Particle's position and orientation with respect to the interface are simultaneously measured as a function of time. From the measured mean square displacement, five particle mobilities (3 translational and 2 rotational) and two translational-rotational cross-correlations are extracted. The fluid dynamics governing equations are solved by the finite element method to numerically evaluate the same mobilities, imposing either slip and no-slip boundary conditions to the flow at the air-water interface. The comparison between experiments and simulations reveals an agreement with no-slip boundary conditions prediction for the translation normal to the interface and the out-of-plane rotation, and with slip ones for parallel translations and in-plane rotation. We rationalize these evidences in the framework of surface incompressibility at the interface.

Low Surface Potential with Glycoconjugates Determines Insect Cell Adhesion at Room Temperature.

Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.

The physical properties of the stick insect pad secretion are independent of body size.

Many insects use adhesive organs to climb. The ability to cling to surfaces is advantageous but is increasingly challenged as animals grow, due to the associated reduction in surface-to-volume ratio. Previous work has demonstrated that some climbing animals overcome this scaling problem by systematically altering the maximum force per area that their adhesive pads can sustain; their adhesive organs become more efficient as they grow, an observation which is also of substantial relevance for the design of bioinspired adhesives. What is the origin of this change in efficiency? In insects, adhesive contact is mediated by a thin film of a liquid, thought to increase adhesive performance via capillary and viscous forces. Here, we use interference reflection microscopy and dewetting experiments to measure the contact angle and dewetting speed of the secretion of pre-tarsal adhesive pads of Indian stick insects, varying in mass by over two orders of magnitude. Neither contact angle nor dewetting speed change significantly with body mass, suggesting that the key physical properties of the pad secretion-its surface tension and viscosity-are size-invariant. Thus, the observed change in pad efficiency is unlikely to arise from systematic changes of the physical properties of the pad secretion; the functional role of the secretion remains unclear.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.