Nature Protocols最新文献

The goal of this protocol is to improve the characterization and performance standardization of multiphoton microscopy hardware across a large user base. We purposefully focus on hardware and only briefly touch on software and data analysis routines where relevant. Here we cover the measurement and quantification of laser power, pulse width optimization, field of view, resolution and photomultiplier tube performance. The intended audience is scientists with little expertise in optics who either build or use multiphoton microscopes in their laboratories. They can use our procedures to test whether their multiphoton microscope performs well and produces consistent data over the lifetime of their system. Individual procedures are designed to take 1-2 h to complete without the use of expensive equipment. The procedures listed here help standardize the microscopes and facilitate the reproducibility of data across setups.

Quantum dots (QDs) exhibit fluorescence properties with promising prospects for biomedical applications; however, the QDs synthesized in organic solvents shows poor biocompatibility, limiting their use in biological systems. We developed an approach for synthesizing QDs in live cells by coupling a series of intracellular metabolic pathways in a precise spatial and temporal sequence. We have validated this approach in yeast (Saccharomyces cerevisiae), Staphylococcus aureus, Michigan Cancer Foundation-7 (MCF-7) and Madin-Darby canine kidney (MDCK) cells. The intracellularly synthesized QDs are inherently stable and biocompatible, making them suitable for the direct in situ labeling of cells and cell-derived vesicles. Here, we provide an optimized workflow for the live-cell synthesis of QDs by using S. cerevisiae, S. aureus or MCF-7 cells. In addition, we detail a cell-free aqueous synthetic system (quasi-biosynthesis) containing enzymes, electrolytes, peptides and coenzymes, which closely mimics the intracellular synthetic conditions used in our cell culture system. In this solution, we synthesize biocompatible ultrasmall QDs that are easier to purify and characterize than those synthesized in cells. The live-cell-synthesized QDs can be used for bioimaging and microvesicle detection, whereas the quasi-biosynthesized QDs are suitable for applications such as biodetection, biolabeling and real-time imaging. The procedure can be completed in 3-4 d for live-cell QD synthesis and 2 h for the quasi-biosynthesis of QDs. The procedure is suitable for users with expertise in chemistry, biology, materials science and synthetic biology. This approach encourages interested researchers to engage in the field of QDs and develop further biomedical applications.

In the mammalian brain, a large network of excitable and modulatory cells efficiently processes, analyzes and stores vast amounts of information. The brain's anatomy influences the flow of neural information between neurons and glia, from which all thought, emotion and action arises. Consequently, one of the grand challenges in neuroscience is to uncover the finest structural details of the brain in the context of its overall architecture. Recent developments in microscopy and biosensors have enabled the investigation of brain microstructure and function with unprecedented specificity and resolution, dendritic spines being an exemplary case, which has provided deep insights into neuronal mechanisms of higher brain function, such as learning and memory. As diffraction-limited light microscopy methods cannot resolve the fine details of brain cells (the 'anatomical ground truth'), electron microscopy is used instead to contextualize functional signals. This approach can be quite unsatisfying given the fragility and dynamic nature of the structures under investigation. We have recently developed a method for combining super-resolution stimulated emission depletion microscopy with functional measurements in brain slices, offering nanoscale resolution in functioning brain structures. We describe how to concurrently perform morphological and functional imaging with a confocal STED microscope. Specifically, the procedure guides the user on how to record astrocytic Ca²⁺ signals at tripartite synapses, outlining a framework for analyzing structure-function relationships of brain cells at nanoscale resolution. The imaging requires 2-3 h and the image analysis between 2 h and 2 d.

The systematic cultivation of species of photosynthetically active 'green' microorganisms in research labs started in the 1940s. Among these microorganisms, Chlamydomonas represents a genus of green biciliated microalgae, of which Chlamydomonas reinhardtii has become the main describing species. For decades C. reinhardtii has been used as an established model organism in biology, including research areas such as molecular biology of eukaryotes, photosynthesis, light receptors, cell metabolism, the dynamics of microtubule assembly and protein transport along cilia. More recently, the use of suspensions of light-responsive living microorganisms has seen a major expansion from the life sciences to the biophysics, statistical physics, fluid dynamics and bioengineering communities. Studies that substantially advance the state of the art in these research areas require the reliable preparation and maintenance of viable, monodisperse and synchronous cell cultures. Although some technical aspects are shared with standard procedures in cell biology and microbiology, Chlamydomonas and its relatives are photosensitive and, simultaneously, motile, meaning this microorganism requires tailored cultivation protocols that are specific to this species. Here we provide guidance on which Chlamydomonas wild-type and mutant strains are suitable for specific experiments and provide detailed step-by-step procedures to measure culture synchronicity, growth rate of the population, average cell size and motility features. The reliable preparation of cell cultures may facilitate future interdisciplinary research using living suspensions of photoactive microorganisms.

Technical lignins are an industrial byproduct of plant biomass processing, for example, paper production or biorefinery operations. They are highly functional and aromatic, making them potentially suitable for a diverse range of applications; however, their exact structural composition depends on the plant species and the industrial process involved. A major bottleneck to lignin valorization and to biorefining in general is the equipment and time investment required for the full characterization of each sample. An array of wet chemical, spectroscopic, chromatographic and thermal methods are typically required to effectively characterize a given lignin sample. To ease the analytical burden, measured lignin properties can be correlated with detailed spectroscopic data obtained from a rapid analytical technique, such as attenuated total reflectance (ATR) Fourier-transform infrared (IR) spectroscopy, which requires minimal sample preparation. With sufficient sensitivity of the spectroscopic data, partial least squares regression models can be calibrated and, thus, predict these properties for future samples for which only the ATR-IR spectra are recorded. So far, several structural and macromolecular properties of lignin have been correlated with ATR-IR spectral data and quantitatively predicted in such a manner, including molecular weight, hydroxyl group content ([OH]), interunit linkage abundance and glass transition temperature. The protocol to apply this powerful lignin characterization methodology is described herein. Here, we also present a simple calibration transfer step, which when implemented before partial least squares regression, addresses the problem of instrument dependency. With the calibrated model, it is possible to determine lignin properties from a single ATR-IR spectral measurement (in ~5 min per sample).

mRNA-based therapies have emerged as a cutting-edge approach for diverse therapeutic applications. However, substantial barriers exist that hinder scientists from entering this research field, including the technical complexity and multiple potential workflows available for formulating and evaluating mRNA lipid nanoparticles (LNPs). Here we present an easy-to-follow and step-by-step guide for mRNA LNP formulation, characterization and in vitro and in vivo evaluation that could lower these barriers, facilitating entry for scientists in academia, industry and clinical settings into this research space. In this protocol, we detail steps for formulating representative mRNA LNPs (0.5 d) and characterizing key parameters (1-6 d) such as size, polydispersity index, zeta potential, mRNA concentration, mRNA encapsulation efficiency and stability. Then, we describe in vitro evaluations (3-6 d), such as protein expression, cell uptake and mechanism investigations (3-5 d), including endosomal escape, as well as in vivo delivery evaluation (2-3 d) encompassing intracellular and secreted protein expression levels, biodistribution and additional tolerability studies (1-2 weeks). Unlike some alternative protocols that may focus on discrete aspects of the workflow-such as formulation, characterization or evaluation-our protocol instead aims to integrate each of these aspects into a simplified, singular workflow applicable across multiple types of mRNA LNP formulations. In describing these procedures, we wish to disseminate one potential workflow for mRNA LNP production and evaluation, with the ultimate goal of furthering innovation, collaboration and the translational advancement of mRNA LNPs.

Protocell research offers diverse opportunities to understand cellular processes and the foundations of life and holds attractive potential applications across various fields. However, it is still a formidable task to construct a true-to-life synthetic cell with high organizational and functional complexity. Here we present a protocol for constructing bacteriogenic protocells by employing prokaryotes as on-site repositories of compositional, functional and structural building blocks to address this challenge. This approach is based on the capture and processing of two spatially segregated bacterial colonies within individual coacervate microdroplets to produce membrane-bounded, molecularly crowded, compositionally, structurally and functionally complex synthetic cells. The bacteriogenic protocells inherit sufficient biological components from their bacterial building units to exhibit highly integrated life-like properties, including biocatalysis, glycolysis and gene expression. The protocells can be endogenously remodeled to acquire diverse proto-organelles including a spatially partitioned nucleus-like DNA/histone-based condensate to store genetic material, membrane-bounded water vacuoles to adjust cellular osmotic pressure, a three-dimensional network of F-actin proto-cytoskeleton to support structural stability and proto-mitochondria to generate endogenous ATP as source of energy. The protocells ultimately develop a nonspherical morphology due to the continuous biogeneration of metabolic products by implanted living bacteria cells. This protocol provides a novel living material assembly strategy for the construction of functional protoliving microdevices and offers opportunities for potential applications in engineered synthetic biology and biomedicine. The protocol takes ~27 d to complete and requires expertise in microbiology, phase separation, biochemistry and molecular biology related techniques.

Ligand-receptor interactions constitute a fundamental mechanism of cell-cell communication and signaling. NicheNet is a well-established computational tool that infers ligand-receptor interactions that potentially regulate gene expression changes in receiver cell populations. Whereas the original publication delves into the algorithm and validation, this paper describes a best practices workflow cultivated over four years of experience and user feedback. Starting from the input single-cell expression matrix, we describe a 'sender-agnostic' approach that considers ligands from the entire microenvironment and a 'sender-focused' approach that considers ligands only from cell populations of interest. As output, users will obtain a list of prioritized ligands and their potential target genes, along with multiple visualizations. We include further developments made in NicheNet v2, in which we have updated the data sources and implemented a downstream procedure for prioritizing cell type-specific ligand-receptor pairs. Although a standard NicheNet analysis takes <10 min to run, users often invest additional time in making decisions about the approach and parameters that best suit their biological question. This paper serves to aid in this decision-making process by describing the most appropriate workflow for common experimental designs like case-control and cell-differentiation studies. Finally, in addition to the step-by-step description of the code, we also provide wrapper functions that enable the analysis to be run in one line of code, thus tailoring the workflow to users at all levels of computational proficiency.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.

Untargeted metabolomics is evolving into a field of big data science. There is a growing interest within the metabolomics community in mining tandem mass spectrometry (MS/MS)-based data from public repositories. In traditional untargeted metabolomics, samples to address a predefined question are collected and liquid chromatography with MS/MS data are generated. We then identify metabolites associated with a phenotype (for example, disease versus healthy) and elucidate or validate their structural details (for example, molecular formula, structural classification, substructure or complete structural annotation or identification). In reverse metabolomics, we start with MS/MS spectra for known or unknown molecules. These spectra are used as search terms to search public data repositories to discover phenotype-relevant information such as organ/biofluid distribution, disease condition, intervention status (for example, pre- and postintervention), organisms (for example, mammals versus others), geography and any other biologically relevant associations. Here we guide the reader through a four-part process: (1) obtaining the MS/MS spectra of interest (Universal Spectrum Identifier) and (2) Mass Spectrometry Search Tool searches to find the files associated with the MS/MS that are in available databases, (3) using the Reanalysis Data User Interface framework to link the files with their metadata and (4) validating the observations. Parts 1-3 could take from hours to days depending on the method used for collecting MS/MS spectra. For example, we use MS/MS spectra from three small molecules: phenylalanine-cholic acid (a microbially conjugated bile acid), phenylalanine-C4:0 and histidine-C4:0 (two N-acyl amides). We leverage the Global Natural Products Social Molecular Networking-based framework to explore the microbial producers of these molecules and their associations with health conditions and organ distributions in humans and rodents.