Biophysics

The relative ease of genetic manipulation in S. cerevisiae is one of its greatest strengths as a model eukaryotic organism. Researchers have leveraged this quality of the budding yeast to study the effects of a variety of genetic perturbations, such as deletion or overexpression, in a high-throughput manner. This has been accomplished by producing a number of strain libraries that can contain hundreds or even thousands of distinct yeast strains with unique genetic alterations. While these strategies have led to enormous increases in our understanding of the functions and roles that genes play within cells, the techniques used to screen genetically modified libraries of yeast strains typically rely on plate or sequencing-based assays that make it difficult to analyze gene expression changes over time. Microfluidic devices, combined with fluorescence microscopy, can allow gene expression dynamics of different strains to be captured in a continuous culture environment; however, these approaches often have significantly lower throughput compared to traditional techniques. To address these limitations, we have developed a microfluidic platform that uses an array pinning robot to allow for up to 48 different yeast strains to be transferred onto a single device. Here, we detail a validated methodology for constructing and setting up this microfluidic device, starting with the photolithography steps for constructing the wafer, then the soft lithography steps for making polydimethylsiloxane (PDMS) microfluidic devices, and finally the robotic arraying of strains onto the device for experiments. We have applied this device for dynamic screens of a protein aggregation library; however, this methodology has the potential to enable complex and dynamic screens of yeast libraries for a wide range of applications.

Key features

• Major steps of this protocol require access to specialized equipment (i.e., microfabrication tools typically found in a cleanroom facility and an array pinning robot).

• Construction of microfluidic devices with multiple different feature heights using photolithography and soft lithography with PDMS.

• Robotic spotting of up to 48 different yeast strains onto microfluidic devices.

Device-induced thrombosis remains a major complication of extracorporeal life support (ECLS). To more thoroughly understand how blood components interact with the artificial surfaces of ECLS circuit components, assessment of clot deposition on these surfaces following clinical use is urgently needed. Scanning electron microscopy (SEM), which produces high-resolution images at nanoscale level, allows visualization and characterization of thrombotic deposits on ECLS circuitry. However, methodologies to increase the quantifiability of SEM analysis of ECLS circuit components have yet to be applied clinically. To address these issues, we developed a protocol to quantify clot deposition on ECLS membrane oxygenator gas transfer fiber sheets through digital and SEM imaging techniques. In this study, ECLS membrane oxygenator fiber sheets were obtained, fixed, and imaged after use. Following a standardized process, the percentage of clot deposition on both digital images and SEM images was quantified using ImageJ through blind reviews. The interrater reliability of quantitative analysis among reviewers was evaluated. Although this protocol focused on the analysis of ECLS membrane oxygenators, it is also adaptable to other components of the ECLS circuits such as catheters and tubing.

Key features

• Quantitative analysis of clot deposition using digital and scanning electron microscopy (SEM) techniques

• High-resolution images at nanoscale level

• Extracorporeal life support (ECLS) devices

• Membrane oxygenators

• Blood-contacting surfaces

Graphical overview

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue’s vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2–3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies.

Aptamers have been selected with strong affinity and high selectivity for a wide range of targets, as recently highlighted by the development of aptamer-based sensors that can differentiate infectious from non-infectious viruses, including human adenovirus and SARS-CoV-2. Accurate determination of the binding affinity between the DNA aptamers and their viral targets is the first step to understanding the molecular recognition of viral particles and the potential uses of aptamers in various diagnostics and therapeutic applications. Here, we describe protocols to obtain the binding curve of the DNA aptamers to SARS-CoV-2 using Enzyme-Linked Oligonucleotide Assay (ELONA) and MicroScale Thermophoresis (MST). These methods allow for the determination of the binding affinity of the aptamer to the infectious SARS-CoV-2 and the selectivity of this aptamer against the same SARS-CoV-2 that has been rendered non-infectious by UV inactivation, and other viruses. Compared to other techniques like Electrophoretic Mobility Shift Assay (EMSA), Surface Plasmon Resonance (SPR), and Isothermal Titration Calorimetry (ITC), these methods have advantages for working with larger particles like viruses and with samples that require biosafety level 2 facilities.

When performing renal biopsy, it is necessary to identify the cortex, where glomeruli are exclusively distributed, to ensure the quality of the specimen for histological diagnosis. However, conventional methods using a stereomicroscope or magnifying lens often fail to clarify the quality of the specimen. We have established a fluorescent-based imaging technique for the on-site assessment of renal biopsy specimens. The fluorescent images can be easily obtained by adding an optical filter to the microscope and with a short incubation of an activatable fluorescent probe. This novel imaging technique can be applied to renal biopsy specimens for distinguishing the renal cortex.

A multitude of membrane-localized receptors are utilized by cells to integrate both biochemical and physical signals from their microenvironment. The clustering of membrane receptors is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we describe a method to fabricate multicomponent, ligand-functionalized microarrays, for spatially segregated and simultaneous monitoring of receptor activation and signaling in individual living cells. While existing micropatterning techniques allow for the display of fixed ligands, this protocol uniquely allows for functionalization of both mobile membrane corrals and immobile polymers with selective ligands, as well as microscopic monitoring of cognate receptor activation at the cell membrane interface. This protocol has been developed to study the effects of clustering on EphA2 signaling transduction. It is potentially applicable to multiple cell signaling systems, or microbe/host interactions.

Graphical abstract:

A side-by-side comparison of clustered or non-clustered EphA2 receptor signaling in a single cell.

The endothelial cells from the microvasculature are key drivers and targets of inflammatory and thrombotic processes in microvascular diseases. The study of bioactive lipids in inflammatory processes has been largely based on two-dimensional endothelial cell cultures. Three-dimensional microvessels-on-a-chip provides an opportunity to monitor the inflammatory phenotype of human microvessels in a more physiological-relevant environment. This protocol describes the culture of endothelial cells as three-dimensional microvessels in the OrganoPlate. The microvessels are treated with tumor necrosis factor alpha to induce inflammation. The collection of samples from the microvessels is optimized for measuring bioactive lipids with liquid chromatography-mass spectrometry, providing a more informative metabolic readout as compared with functional assays.

The design of effective nanoformulations that target metastatic breast cancers is challenging due to a lack of competent imaging and image analysis protocols that can capture the interactions between the injected nanoparticles and metastatic lesions. Here, we describe the integration of in vivo whole-body PET-CT with high temporal resolution, ex vivo whole-organ optical imaging and high spatial resolution confocal microscopy to deconstruct the trafficking of injectable nanoparticle generators encapsulated with polymeric doxorubicin (iNPG-pDox) in pulmonary metastases of triple-negative breast cancer. We describe the details of image acquisition and analysis in a step-wise manner along with the development of a mouse model for metastatic breast cancer. The methods described herein can be easily adapted to any nanoparticle or disease model, allowing a standardized pipeline for in vivo preclinical studies that focus on delineating nanoparticle kinetics and interactions within metastases.

Biohybrid robotics is a growing field that incorporates both live tissues and engineered materials to build robots that address current limitations in robots, including high power consumption and low damage tolerance. One approach is to use microelectronics to enhance whole organisms, which has previously been achieved to control the locomotion of insects. However, the robotic control of jellyfish swimming offers additional advantages, with the potential to become a new ocean monitoring tool in conjunction with existing technologies. Here, we delineate protocols to build a self-contained swim controller using commercially available microelectronics, embed the device into live jellyfish, and calculate vertical swimming speeds in both laboratory conditions and coastal waters. Using these methods, we previously demonstrated enhanced swimming speeds up to threefold, compared to natural jellyfish swimming, in laboratory and in situ experiments. These results offered insights into both designing low-power robots and probing the structure-function of basal organisms. Future iterations of these biohybrid robotic jellyfish could be used for practical applications in ocean monitoring.

The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two fluorescent proteins are in close proximity. Combining FRET analysis with membrane permeabilization, the contribution of small molecules such as nucleotides to the conformational change can be evaluated in living cells.