Biophysics

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.

Traditional methods for studying protein–protein interactions often lack the resolution to quantitatively distinguish distinct oligomeric states, particularly for membrane proteins within their native lipid environments. To address this limitation, we developed SiMPull-POP (single-molecule pull-down polymeric nanodisc photobleaching), a single-molecule technique designed to quantify membrane protein oligomerization with high sensitivity and in a near-native context. The goal of SiMPull-POP is to enable precise, quantitative analysis of membrane protein assembly by preserving native lipid interactions using diisobutylene maleic acid (DIBMA) to form nanodiscs. Unlike ensemble methods such as co-immunoprecipitation or FRET, which average out heterogeneous populations, SiMPull-POP uses photobleaching to resolve monomeric, dimeric, and higher-order oligomeric states at the single-molecule level. We validated SiMPull-POP using several model systems. A truncated, single-pass transmembrane protein (Omp25) appeared primarily monomeric, while a membrane-tethered FKBP protein exhibited ligand-dependent dimerization upon addition of the AP ligand. Applying SiMPull-POP to EphA2, a receptor tyrosine kinase, we found it to be mostly monomeric in the absence of its ligand, Ephrin-A1, and shifting toward higher-order oligomers upon ligand binding. To explore factors influencing ligand-independent assembly, we modulated membrane cholesterol content. Reducing cholesterol induced spontaneous EphA2 oligomerization, indicating that cholesterol suppresses receptor self-association. Overall, SiMPull-POP offers significant advantages over conventional techniques by enabling quantitative, single-molecule resolution of membrane protein complexes in a native-like environment. This approach provides critical insights into how membrane properties and external stimuli regulate protein assembly, supporting broader efforts to understand membrane protein function in both normal and disease states.

Autonomic regulation of heart and respiratory rates is essential for understanding brain–body interactions in health and disease. Preclinical cardiovascular recordings are often performed under anesthesia or via telemetry, both of which introduce physiological confounds such as stress or impaired recovery due to the need for acute or chronic implantation of sensors. Here, we present a minimally invasive protocol for simultaneous acquisition of high-quality electrocardiography and respiratory signals in awake mice. Using an in-house-modified physiological monitor in awake, head-fixed mice that were briefly habituated to experimental conditions, we ultimately enable stable, long-term physiological recordings alongside in vivo microscopy. This protocol provides a robust, low-stress method for acquiring physiological signals, enabling the simultaneous study of cardiovascular–cerebral dynamics in awake head-fixed mice, thereby enhancing the translational relevance of preclinical measurements.

Conventional Schlieren optics equipment typically operates on a large optical table, which is inconvenient for imaging small samples or thin layers of transparent materials. We describe an imaging device based on Schlieren optics, aided by a slight shift in light reflected from two surfaces. The device is designed to place the sample between a thick concave mirror and a camera next to a point-light source located at the spherical origin of the concave mirror. The compact device is portable and convenient. It is similarly capable of sensitively detecting patterns in gaseous or liquid media created by a density gradient when the optical effect is too subtle to be detectable by regular cameras and scanners. The new device is particularly suitable for detecting translucent samples, including thin fluid films on the order of micrometers, tissue slices, and other biological samples. We show two examples of how our device can be applied to imaging biological samples. The first compares images acquired using several techniques of a bacterial swarm spread over an agar plate; the second is a set of images of human cells grown on a tissue culture plate.