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Past Issue in 2025
Volume: 15, Issue: 6
Biological Engineering
Dual-Modal Fast Photoacoustic/Ultrasound Localization Imaging with Sparsity-Constrained Optimization
Dual-modal imaging, combining photoacoustic (PA) and ultrasound localization (UL) with microbubbles, holds substantial promise across biomedical fields such as oncology, neuroscience, nephrology, and immunology. The combination of PA and UL imaging faces challenges due to acquisition speed mismatches, limiting their combined efficacy. Here, we introduce a protocol that applies sparsity constraint optimization to accelerate dual-modal data acquisition, enabling in vivo super-resolution imaging of vascular and physiological structures at under two seconds per frame. The protocol provides detailed guidelines for constructing an interleaved PA/UL (PAUL) imaging system, covering material selection, system setup, and calibration, as well as methods for image acquisition, reconstruction, post-processing, and troubleshooting. This approach empowers the biomedical community to establish a rapid, dual-modal PAUL imaging platform, broadening biomedical applications and advancing imaging capabilities in clinical research.
Cell Biology
Generation of FECD Phenotypes in the Mouse Cornea by UVA Exposure and Surgical Removal of its Corneal Endothelial Layer
Fuchs endothelial corneal dystrophy (FECD) is a rare and multifactorial disorder leading to cell death in the innermost layer of the cornea, i.e., the endothelium; UV radiation is reported as the major environmental risk for the disease. Establishing an animal model for this disease has remained challenging in FECD research. We have developed a detailed protocol for the establishment of a UVA-induced FECD mouse model and removal of corneal endothelium from the eye for further molecular and histological studies by taking references from previous studies. UVA light of 500 J/cm2 was focused on the C57BL/6J female mouse cornea and kept for an observation period of 90 days. The animal developed corneal scarring by the end of three months. Slit-lamp microscopy and alizarin red–trypan blue staining confirmed endothelial cell death and formation of corneal guttae in the endothelium. Surgical removal of the endothelial layer was successfully done in the diseased mouse, and the result was confirmed by immunofluorescence. This study is relevant for in-depth research using a FECD mouse model, which will surpass the limitation of human tissue scarcity and can be used for in vivo drug targeting to develop therapeutics to cure FECD.
Developmental Biology
Visualization of mRNA Translation Within Germ Granule Biphasic Organization in Drosophila Early Embryo
Super-resolution imaging of RNA–protein (RNP) condensates has shown that most are composed of different immiscible phases reflected by a heterogenous distribution of their main components. Linking RNA–protein condensate’s inner organization with their different functions in mRNA regulation remains a challenge, particularly in multicellular organisms. Drosophila germ granules are a model of RNA–protein condensates known for their role in mRNA storage and localized protein production in the early embryo. Present at the posterior pole of the embryo within a specialized cytoplasm called germplasm, they are composed of maternal mRNAs as well as four main proteins that play a key role in germ granule formation, maintenance, and function. Germ granules are necessary and sufficient to drive germ cell formation through translational regulation of maternal mRNAs such as nanos. Due to their localization at the posterior tip of the ovoid embryo and small size, the classical imaging setup does not provide enough resolution to reach their inner organization. Here, we present a specific mounting design that reduces the distance between the germ granule and the objectives. This method provides optimal resolution for the imaging of germ granules by super-resolution microscopy, allowing us to demonstrate their biphasic organization characterized by the enrichment of the four main proteins in the outermost part of the granule. Furthermore, combined with the direct visualization of nanos mRNA translation using the Suntag approach, this method enables the localization of translation events within the germ granule’s inner organization and thus reveals the spatial organization of its functions. This approach reveals how germ granules serve simultaneously as mRNA storage hubs and sites of translation activation during development. This work also highlights the importance of considering condensates’ inner organization when investigating their functions.
Microbiology
Improved Extraction Methods to Isolate High Molecular Weight DNA From Magnaporthaceae and Other Grass Root Fungi for Long-Read Whole Genome Sequencing
This manuscript details two modified protocols for the isolation of long-stranded or high molecular weight (HMW) DNA from Magnaporthaceae (Ascomycota) fungal mycelium intended for whole genome sequencing. The Cytiva Nucleon PhytoPure and the Macherey-Nagel NucleoBond HMW DNA kits were selected because the former requires lower amounts of starting material and the latter utilizes gentler methods to maximize DNA length, albeit at a higher requirement for input material. The Cytiva Nucleon PhytoPure kit successfully recovered HMW DNA for half of our fungal species by increasing the amount of RNase A treatment and adding in a proteinase K treatment. To reduce the impact of pigmentation development, which occurs toward later stages of culturing, extractions were run in quadruplicate to increase overall DNA concentration. We also adapted the Macherey-Nagel NucleoBond HMW DNA kit for high-quality HMW DNA by grinding the sample to a fine powder, overnight lysis, and splitting the sample before washing the precipitated DNA. For both kits, precipitated DNA was spooled out pre-washing, ensuring a higher percentage of high-integrity long strands. The Macherey-Nagel protocol offers advantages over the first through the utilization of gravity columns that provide gentler treatment, yielding >50% of high-purity DNA strands exceeding 40 kbp. The limitation of this method is the requirement for a large quantity of starting material (1 g). By triaging samples based on the rate of growth relative to the accumulation of secondary metabolites, our methodologies hold promise for yielding reliable and high-quality HMW DNA from a variety of fungal samples, improving sequencing outcomes.
HS–GC–MS Method for the Diagnosis of IBD Dynamics in a Model of DSS-Induced Colitis
Inflammatory bowel disease (IBD) is highly prevalent globally and, in the majority of cases, remains asymptomatic during its initial stages. The gastrointestinal microbiota secretes volatile organic compounds (VOCs), and their composition alters in IBD. The examination of VOCs could prove beneficial in complementing diagnostic techniques to facilitate the early identification of IBD risk. In this protocol, a model of sodium dextran sulfate (DSS)-induced colitis in rats was successfully implemented for the non-invasive metabolomic assessment of different stages of inflammation. Headspace–gas chromatography–mass spectrometry (HS–GC–MS) was used as a non-invasive method for inflammation assessment at early and remission stages. The disease activity index (DAI) and histological method were employed to assess intestinal inflammation. The HS–GC–MS method demonstrated high sensitivity to intestine inflammation, confirmed by DAI and histology assay, in the acute and remission stages, identifying changes in the relative content of VOCs in stools. HS–GC–MS may be a useful and non-invasive method for IBD diagnostics and therapy effectiveness control.
Differentiation of Bacillus cereus Species Based on Detected Unamplified Bacterial 16S rRNA by DNA Nanomachine
Traditional approaches for the detection and differentiation of Bacillus cereus group species often face challenges due to the complexity of genetic discrimination between species. In this protocol, we propose a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay incorporates a universal fluorescent reporter and four DNA binding fragments, three of which are responsible for “opening up” the folded rRNA while the fourth strand is responsible for detecting single nucleotide variation (SNV) with high selectivity. The binding of the DNM to 16S rRNA results in the formation of the 10-23 DNAzyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. The developed biplex assay enables the detection of B. thuringiensis 16S rRNA and B. mycoides at fluorescein and Cy5 channels, respectively. The protocol offers two detection options: one utilizing extracted total RNA and the other involving crude cell lysate. The latter enables a fast and straightforward detection after 1.5 h with a hands-on time of ~15 min. The new protocol may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis.
Evaluating In Vivo Translation Inhibition via Puromycin Labeling in Botrytis cinerea Germlings Treated With Antifungal Peptides
Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen–host interactions in filamentous fungi.
A Highly Sensitive and Selective DAMP Assay for the Detection of Bacterial Pathogens Associated With Brain Inflammation
The early detection of meningitis pathogens—including Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Klebsiella pneumoniae—through point-of-care (POC) systems is essential for mitigating the risk of neurological damage, enhancing patient outcomes, and facilitating prompt clinical decision-making. Nucleic acid amplification testing (NAAT) is a promising tool for improving the diagnosis process of bacterial pathogens associated with brain inflammation. This is due to its high sensitivity, rapidity, and compatibility with portable diagnostic platforms, making it particularly suitable for POC applications. This protocol introduces an innovative diagnostic approach designed to function effectively without the need for advanced laboratory equipment. By leveraging dual-priming isothermal amplification (DAMP), the assay uses custom internal primers to enhance specificity and minimize false results. Brilliant Green is used in this assay for fluorescence detection due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay demonstrated excellent specificity, absence of false positives, sensitivity comparable to loop-mediated isothermal amplification (LAMP), and a high S/B ratio.
Molecular Biology
Sensitive and Adaptable Turn-On Maturation (ATOM) Fluorescent Biosensors for Detecting Subcellular Localization of Protein Targets in Cells
Fluorescent protein biosensors (FPBs) that turn on—go from dark to bright upon binding their ligands—enable the detection of targets in living cells with high sensitivity and spatial localization. Several approaches exist for creating turn-on FPBs, most notably the method that gave rise to the GCaMP family of genetically encoded calcium indicators. However, it remains challenging to modify these sensors to recognize new ligands. We recently developed adaptable turn-on maturation (ATOM) biosensors, in which target recognition by a small binding domain triggers chromophore maturation in the fluorescent protein to which it is attached. ATOM sensors are advantageous because they are generalizable (by virtue of the monobody and nanobody binding domains) and modular (binding domains and fluorescent proteins of various colors can be mixed and matched for multiplexed imaging), capable of detecting endogenously expressed proteins, and able to function in subcellular compartments including the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria. The protocols herein detail how to design, clone, and screen new ATOM sensors for detecting targets of choice. The starting materials are the genes encoding for a monobody or nanobody and for a cyan, yellow, or red fluorescent protein. We also present general guidelines for creating ATOM sensors using binding domains other than nanobodies and monobodies.
Neuroscience
Developing a Ministroke Model in Mouse Barrel Cortex
Stroke is a worldwide leading cause of death and long-term disability, with ischemic strokes making up approximately 85% of all cases. There is a significant need for an ideal animal model that accurately replicates the disease’s pathology to study the molecular mechanisms of brain injury. Various experimental models have been created to induce middle cerebral artery occlusion (MCAO), including intraluminal MCAO, photothrombotic models, endothelin-1 injections, and electrocoagulation. However, these often result in large infarct or lesion volumes accompanied by considerable variability. In this study, we present a ministroke model that specifically targets the mouse barrel cortex, making it suitable for investigating the mechanisms of minor strokes and stroke recurrence. In our model, the distal branch of the right middle cerebral artery (MCA), which supplies the sensorimotor cortex, is permanently ligated using 10-0 sutures. This is followed by a 7-min occlusion of the bilateral common carotid arteries (CCAs) and subsequent reperfusion. This approach produces a mild stroke characterized by small and consistent lesion volumes and very low mortality rates. A well-trained experimenter can achieve nearly zero mortality with this technique. Furthermore, this model of localized ischemia induces lesions in the functionally defined barrel cortex, allowing the use of the vibrissae-evoked forelimb placing test to assess functional outcomes.
Time-Lapse Super-Resolution Imaging and Optical Manipulation of Growth Cones in Elongating Axons and Migrating Neurons
The growth cone is a highly motile tip structure that guides axonal elongation and directionality in differentiating neurons. Migrating immature neurons also exhibit a growth cone–like structure (GCLS) at the tip of the leading process. However, it remains unknown whether the GCLS in migrating immature neurons shares the morphological and molecular features of axonal growth cones and can thus be considered equivalent to them. Here, we describe a detailed method for time-lapse imaging and optical manipulation of growth cones using a super-resolution laser-scanning microscope. To observe growth cones in elongating axons and migrating neurons, embryonic cortical neurons and neonatal ventricular–subventricular zone (V-SVZ)-derived neurons, respectively, were transfected with plasmids encoding fluorescent protein–conjugated cytoskeletal probes and three-dimensionally cultured in Matrigel, which mimics the in vivo background. At 2–5 days in vitro, the morphology and dynamics of these growth cones and their associated cytoskeletal molecules were assessed by time-lapse super-resolution imaging. The use of photoswitchable cytoskeletal inhibitors, which can be reversibly and precisely controlled by laser illumination at two different wavelengths, revealed the spatiotemporal regulatory machinery and functional significance of growth cones in neuronal migration. Furthermore, machine learning–based methods enabled us to automatically segment growth cone morphology from elongating axons and the leading process. This protocol provides a cutting-edge methodology for studying the growth cone in developmental and regenerative neuroscience, being adaptable for various cell biology and imaging applications.
Plant Science
Protocol for Inoculation of PGPR Staphylococcus sciuri to Seeds and Seedlings of Rice and Tomato Plants for Increased Root and Shoot Growth
Plant growth–promoting rhizobacteria (PGPR) can be used as biofertilizers to enhance crop growth for better yield and soil fertility restoration. PGPR possesses certain traits such as nutrient solubilization, phytohormone production, and production of key enzymes for improved crop growth. These traits are also important for inhibiting the growth of plant root pathogens, improving root development, and conferring stress tolerance. However, the mere presence of PGPR traits in isolated bacteria may not directly reflect an improvement in plant growth, warranting researchers to evaluate phenotypic and physiological changes upon inoculation. The current manuscript provides a detailed step-by-step procedure for inoculating the PGPR Staphylococcus sciuri into seeds and seedlings of rice and tomato plants for visualizing the enhancement of root and shoot growth. The surface-sterilized seeds of rice and tomato plants are inoculated overnight with an actively grown log-phase culture of S. sciuri, and differences in growth and biomass of seedlings that emerged from the inoculated and uninoculated seeds are analyzed 10 days after germination. Plants grown in pots with sterile soil are also treated with PGPR S. sciuri by soil drenching. A remarkable increase in root and shoot growth is observed in inoculated plants. We suggest that treating seeds with bacteria and enriching the soil with bacterial inoculum provides an adequate load of PGPR that facilitates growth improvement. This method can be a reliable choice for screening and evaluating plant growth promotion by either isolated bacteria or bacterial consortia with plant-beneficial traits.
Systems Biology
PCR-Based Genotyping of Zebrafish Genetic Mutants
Zebrafish genetic mutants have emerged as a valuable model system for studying various aspects of disease and developmental biology. Mutant zebrafish embryos are generally identified based on phenotypic defects at later developmental stages, making it difficult to investigate underlying molecular mechanisms at earlier stages. This protocol presents a PCR-based genotyping method that enables the identification of wild-type, heterozygous, and homozygous zebrafish genetic mutants at any developmental stage, even when they are phenotypically indistinguishable. The approach involves the amplification of specific genomic regions using carefully designed primers, followed by gel electrophoresis. This genotyping method facilitates the investigation of the molecular mechanisms driving phenotypic defects that are observed at later timepoints. This protocol allows researchers to perform analyses such as immunofluorescence, RT-PCR, RNA sequencing, and other molecular experiments on early developmental stages of mutants. The availability of this protocol expands the utility of zebrafish genetic mutants for elucidating the molecular underpinnings of various biological processes throughout development.