Biophysics

Stochastic optical reconstruction microscopy (STORM) is a single-molecule localization microscopy technique that enables visualization of cellular structures beyond the diffraction limit. This approach has revealed previously inaccessible ultrastructural details in a wide range of cellular components, including the actin cytoskeleton, clathrin-coated pits, mitochondria, and bacterial nucleoid-associated proteins. STORM relies on the sequential emission of single photons from photosensitive fluorophores, which are precisely localized before entering a dark state or undergoing photobleaching. By activating fluorophores individually and fitting their point spread functions (PSFs), the center of mass can be calculated with a localization precision of up to ~20 nm. The parallel detection of thousands of single-molecule events, each assigned to distinct spatial coordinates, enables the reconstruction of a high-resolution image. Here, we describe a simple and efficient STORM workflow—including sample preparation, image acquisition, and quality control measurements—that we used to visualize various subcellular structures, such as mitochondria, microtubules, and lysosomes labeled with the commonly employed cyanine dye Alexa Fluor 647, as well as the actin cytoskeleton stained with Alexa Fluor 488–conjugated phalloidin. Image acquisition was performed using a conventional epifluorescence/total internal reflection (TIRF) microscope adapted for STORM imaging. Key adaptations included the use of a 160×/1.43 NA oil-immersion objective and a high-power mode, which concentrates the laser beam onto a small region of the sample, ensuring sufficient light intensity to drive fluorophores into the dark state. In addition, implementing a 1.6× magnification lens and a 4×4 binning camera mode allowed us to achieve a 100-nm pixel size optimal for reliable molecule detection. We believe that this protocol will be highly valuable to the microscopy community, as it lowers technical barriers to performing STORM on widely available microscopy platforms, thereby facilitating broader implementation of this powerful super-resolution technique.

The spatiotemporal dynamics and density of actin networks are key determinants of actin cytoskeleton–mediated cellular functions. In vitro reconstitution systems have been widely used to study actin cytoskeletal dynamics; however, many existing approaches offer limited flexibility in controlling the geometry, thickness, and density of the assembled actin networks. Here, we present an in vitro optogenetic protocol that enables precise control of actin network assembly on supported lipid bilayers using an improved light-induced dimer (iLID)-SspB-based light-inducible dimerization system. In this system, His-mEGFP-iLID is anchored to a Ni-NTA-containing lipid bilayer, while SspB-mScarlet-I-VCA, a nucleation-promoting factor fused with SspB, together with other actin cytoskeletal proteins, is supplied in bulk solution. Upon blue light illumination, SspB-mScarlet-I-VCA is recruited to the membrane in a spatially and temporally defined manner, inducing localized actin polymerization. By tuning illumination patterns and duration, actin networks with defined density, thickness, and geometry can be generated, and polymerization can be rapidly halted by stopping illumination. This protocol provides a versatile platform for reconstructing actin networks with controlled spatial organization and density, enabling quantitative analysis of density-dependent interactions between actin networks and actin-binding proteins.

Among the biophysical techniques used in fragment-based drug discovery (FBDD) campaigns, crystallography is the most sensitive, allowing for the identification of low-affinity ligands and the characterization of protein–ligand complexes at atomic resolution. Although powerful, the proper application of this technique depends on obtaining crystals capable of diffracting X-rays at high resolution. Additionally, in crystallographic compound screening, the crystals must be resistant to multiple organic solvents used in chemical libraries, such as DMSO. In this protocol, we describe recombinant protein production suitable for crystallization and procedures for X-ray crystallographic screening of a library of 768 fragments. As a case study, we used the Schistosoma mansoni thioredoxin glutathione reductase (SmTGR), a redox enzyme with a key role in controlling oxidative stress in parasites of the Schistosoma genus, which causes schistosomiasis. As a validated pharmacological target, SmTGR is used in the development of new schistosomicidal drugs. The experimental pipeline includes SmTGR expression, purification, and crystallization, crystal soaking, diffraction data collection, and refinement. The 768 fragments from the Diamond-SGC Poised Library (DSPL) were individually soaked onto the crystals, and diffraction data were collected and processed at the I04-1 beamline of the Diamond Light Source synchrotron. Diffraction data were subsequently analyzed using PanDDA to identify fragment-binding events and to enable reliable detection of low-occupancy ligands within the protein crystal structures. In addition to the core experimental steps, this protocol incorporates systematic approaches to overcome limitations frequently encountered in crystallographic screening campaigns, including assessment of crystal solvent tolerance, acceleration of crystal mounting through the use of auxiliary devices, acoustic dispensing–based soaking of hundreds of fragments for low material consumption and high throughput, automated data collection, and efficient data analysis pipeline for the detection of weakly bound ligand. This protocol can be broadly applied to screen diverse compound sets against multiple targets amenable to crystallization.

Nanoparticle vaccines can provide advantages over traditional vaccine methodologies, including adjuvant delivery to enhance the effectiveness of recombinant antigens. Many approaches exist to formulate different vaccine nanoparticles, which are designed for different biomolecular cargos, adjuvant compositions, and disease targets. Here, a protocol is described to produce nanoliposomes whose surface is decorated with recombinant protein influenza antigens with monophosphoryl lipid A and QS-21 adjuvants incorporated into the lipid bilayer for protection against influenza infection. This protocol includes methods for producing adjuvanted liposomes and coupling with His-tagged antigens for surface decoration of the particle. This allows for a rapid methodology of producing immunogenic antigen-presenting liposomes that can be tailored to display a combination of influenza surface antigens.

ADGRL4 is an adhesion G protein-coupled receptor (aGPCR) implicated in tumour progression in multiple malignancies. We recently determined the first cryo-EM structure of active-state ADGRL4, revealing its weak coupling to the heterotrimeric G protein G_q and providing insights into its activation mechanism. Here, we describe a complete modular workflow for purifying active-state ADGRL4 over 2–3 days using a multifunctional tagging strategy incorporating multiple orthogonal detection, purification, and cleavage tags at the N-terminus as well as a tethered mini-G_q at the C-terminus. This configuration enhanced receptor cell-surface expression and stability and allowed different purification strategies to be tested during the development of the purification protocol. Although developed and optimised for ADGRL4, this approach is readily transferable to other weakly coupling aGPCRs or GPCRs where complex stability is a limiting factor for structural analysis.

Plasma membrane–associated condensates driven by liquid–liquid phase separation represent a novel mechanism of receptor-mediated signaling transduction, serving as mesoscale platforms that concentrate signaling molecules and modulate reaction kinetics. Condensate formation is a highly dynamic process that occurs within seconds to minutes following receptor activation. Here, we present methods for de novo reconstituting liquid-like condensates on supported lipid bilayers and assessing the condensate fluidity using fluorescence recovery after photobleaching (FRAP). This protocol encompasses supported lipid bilayer preparation, condensation imaging, and FRAP analysis using total internal reflection fluorescence (TIRF) microscopy. Supported lipid bilayers provide a membrane-mimicking environment for receptor signaling cascades, offering mechanistic insights into protein–protein and lipid–protein interactions amid micron-scale condensates. The protocol can also be adapted to study condensates associated with the internal membranes of the Golgi apparatus, mitochondria, and other organelles.

The compound eyes of Drosophila are widely used to gain valuable insights into genetics, developmental biology, cell biology, disease biology, and gene regulation. Various parameters, such as eye size, pigmentation loss, formation of necrotic patches, and disorientation, fusion, or disruption of ommatidial arrays, are commonly assessed to evaluate eye development and degeneration. We developed an improved imaging method named low-angle ring illumination stereomicroscopy (LARIS) to capture high-contrast images of the Drosophila compound eye. Different optical alignments were tested to capture the fly compound eye image under the stereomicroscope; the highest contrast with minimal reflection was achieved through the LARIS method. The images captured using LARIS clearly showed ommatidial fusion, disorientation, and pigmentation loss, which were hardly visible with a conventional imaging method in the degenerating compound eyes of Drosophila. In addition to its research applications, this protocol is cost-effective due to the low expenses associated with supplies and equipment. We anticipate that LARIS will facilitate high-contrast imaging of the compound eyes in Drosophila and other insects.

The extracellular matrix (ECM) critically shapes melanoma progression and therapeutic response, yet commonly used matrices such as Matrigel fail to capture tissue- and disease-specific ECM properties. This protocol provides a streamlined and scalable method for generating murine, tissue-specific ECM hydrogels from skin, lung, and melanoma tumors, therefore overcoming the restricted materials of mouse-derived ECM. The workflow integrates tissue-tailored decellularization, lyophilization, mechanical fragmentation, pepsin digestion, and physiological polymerization to produce hydrogels that reliably preserve fibrillar collagen architecture and organ-specific ECM cues. Decellularization efficiency and ECM integrity are validated by DNA quantification, H&E staining, and Picrosirius Red staining analysis. These hydrogels provide a species- and tissue-matched platform for studying melanoma–ECM–immune interactions, pre-metastatic niche features, and therapy-induced ECM remodeling. Overall, this protocol offers a reproducible and physiologically relevant ECM model that expands experimental capabilities for melanoma biology and treatment-resistance research and that can be easily extended to other tumors and tissues.

Expansion microscopy (ExM) is an innovative and cost-effective super-resolution imaging technique that enables nanoscale visualization of biological structures using conventional fluorescence microscopes. By physically enlarging biological specimens, ExM circumvents the diffraction limit and has become an indispensable tool in cell biology. Ongoing methodological advances have further enhanced its spatial resolution, labeling versatility, and compatibility with diverse sample types. However, ExM imaging is often hindered by sample drift during image acquisition, caused by subtle movements of the expanded hydrogel. This drift can distort three-dimensional reconstruction, compromising both visualization accuracy and quantitative analysis. To overcome this limitation, we developed 3D-Aligner, an advanced and user-friendly image analysis software that computationally corrects sample drift in fluorescence microscopy datasets, including but not limited to those acquired using ExM. The algorithm accurately determines drift trajectories across image stacks by detecting and matching stable background features, enabling nanometer-scale alignment to restore structural fidelity. We demonstrate that 3D-Aligner robustly corrects drift across ExM datasets with varying expansion factors and fluorescent labels. This protocol provides a comprehensive, step-by-step workflow for implementing drift correction in ExM datasets, ensuring reliable three-dimensional imaging and quantitative assessment.

Most viruses extensively remodel their host cells to establish productive infection. Visualization of virus-induced cellular remodeling by electron microscopy (EM) has been revolutionized in recent years by advances in cryo-focused ion beam (cryo-FIB) milling paired with cryo-electron tomography (cryo-ET). As cryo-FIB/ET becomes more widely available, there is a need for beginner-friendly guides to optimize the preparation of virus-infected mammalian cells on EM grids. Here, we provide an in-house protocol for new users for preparing samples of cells infected with herpes simplex virus 1 (HSV-1) for cryo-FIB/ET. This protocol guides users in how to seed infected cells onto grids, blot, and plunge-freeze grids using basic, manual equipment. It also provides tips on how to screen and prioritize grids for efficient milling and data collection.