Neutrophils, important leukocytes of innate immunity, have traditionally been considered a homogeneous cell population. Nevertheless, recent evidence has shown that neutrophils exist in several subpopulations. One such subpopulation is low-density neutrophils (LDN). LDN are found in small numbers in the blood of healthy individuals, but their numbers increase significantly in diseases such as systemic lupus erythematosus, autoimmune disorders, cancer, and infections. In these cases, LDN may participate in the pathogenesis of the disease. The only way to isolate LDN is through density-gradient centrifugation of peripheral blood. However, after centrifugation, LDN co-purify with mononuclear cells. Thus, studying this neutrophil subpopulation is challenging. There is no standard methodology to separate LDN from mononuclear cells. Typically, LDN are separated by cell sorting in a flow cytometer. However, this method requires long sorting times (hours) to obtain enough pure cells for further functional studies. This seriously affects the viability and function of cells. Here, we propose a practical method to obtain large numbers of pure and viable LDN in a short time. After density-gradient centrifugation, the mononuclear cell fraction is incubated with anti-CD66b magnetic microbeads, and then LDN are separated through magnetic columns in < 30 min. Purified LDN (CD66b cells) are labelled with monoclonal antibodies against CD10, CD11b, CD14, CD15, CD16b, CD33, CD62L, CD66b, and CD98, and are analyzed by flow cytometry for confirmation. Purified LDN are completely functional as indicated by their capacity to produce reactive oxygen species, and to form neutrophil extracellular traps. This new purification method results in LDN with high purity (more than 90%) and viability (more than 96%) in a short time period. This method can easily be scaled up to obtain large numbers of pure LDN to evaluate LDN functions in different diseases through biochemical or other omics analysis.

Over the past two decades, piezoelectric materials have become an integral part of structural health monitoring (SHM) and energy harvesting. These materials are available in various forms and configurations, yet the potential of curved configurations remains largely unexplored. This article experimentally explores the performance of singly curved thin piezoelectric transducers compared to their straight configuration when embedded in reinforced concrete (RC) structures for energy harvesting and SHM applications. The research includes a side-by-side comparison of these configurations based on (a) the open-circuit voltage generated by them under pure harmonic excitations, (b) the power generated under the impedance matching conditions, and (c) their potential for power storage in capacitors under real-life erratic vibrations with impedance mismatch conditions. Additionally, the article investigates the damage detection capabilities of curved piezo transducers using the electromechanical impedance (EMI) technique, an area that has not been thoroughly explored. The results from all four experiments demonstrate that the curved piezo transducers outperform their straight configurations in both SHM and energy harvesting applications. Therefore, the findings from these investigations are crucial for the effective utilization of curved piezoelectric transducers in real-world RC structures, enhancing both energy harvesting and structural health monitoring, which in fact, leads to the economic maintenance and safe operation of RC structures.

The ability to detect protein and mRNA in the same assay helps us understand how the cell is priming for an arrival and what cues it receives as it travels and interacts in the environment it finds itself in. Higher-plexed imaging now permits characterization of spatially resolved mRNA (spatial transcriptomics) and protein (spatial proteomics) simultaneously. To avoid redundancy, these analytes can be analyzed in a single tissue section. There are many technologies that allow for many proteins and few mRNAs or the inverse, but as the central acting analytes are proteins, this manuscript is focused on integrating protein imaging mass cytometry (IMC) with mRNA in situ hybridization (ISH) utilizing metal probes. This allows us to understand what messages the cell is priming or is sending rapidly into the world as it communicates in its microenvironment. It also overcomes limitations related to the detection of secreted proteins or challenging markers that are usually hard to quantify and visualize utilizing antibodies only. This protocol also minimizes the need for compensations or post-processing seen in higher plex fluorescent-based methods, as there is no fluorescent spectral spillover in a metal probe-based detection system, nor are there tissue-specific autofluorescence considerations.

Obesity has emerged as a predominant global health epidemic, with nearly half of the world's population now classified as overweight or obese. A key pathological feature in obese individuals is ectopic lipid deposition in non-adipose tissues, including the pancreas-a condition termed pancreatic steatosis or fatty pancreas. In high-fat diet (HFD)-induced rodent models, pancreatic steatosis consistently precedes hepatic steatosis, underscoring the particular susceptibility of the pancreas to lipid accumulation. This early involvement positions the pancreas as a critical organ for understanding metabolic dysregulation in obesity. Although diet-induced obese (DIO) rats recapitulate core aspects of human disease progression, well-characterized animal models that reliably exhibit this pancreatic phenotype remain scarce in the literature. A significant methodological consideration in modeling human obesity is the timing of HFD introduction. Many existing models initiate HFD during the weaning period, which may introduce confounding developmental metabolic programming effects that do not fully reflect human obesity, which primarily develops post-developmentally. In contrast, the present study establishes a standardized protocol in which HFD is introduced post-weaning. This approach more accurately mimics the common human trajectory of obesity onset in adulthood, avoids early-life metabolic adaptations, and results in a more physiologically relevant progression toward pancreatic steatosis. This research provides a detailed and reproducible framework for generating a rat model of obesity characterized by prominent fatty pancreas pathology. The methodology includes a 1-week acclimatization phase, a 14-week HFD induction period starting after weaning, and systematic tissue harvesting. Two tissue processing pathways are described: snap-freezing for molecular analyses (e.g., Western blotting) and paraformaldehyde perfusion-fixation for morphological evaluations (e.g., IHC, H&E). This model offers a robust platform for mechanistic investigations into obesity-associated pancreatic metabolic dysfunction.

The present study employed a combination of behavioral and event-related potential (ERP) measures to investigate the effect of ego depletion on perceptual anticipation in tennis-trained undergraduates under negative emotion. The findings will provide a theoretical foundation for enhancing training efficacy in this specific population. Negative emotion was induced by film clips, while ego-depletion was elicited through a Stroop task. Perceptual anticipation was evaluated using temporally occluded tennis videos. Under negative emotion, participants exhibited shorter reaction time and higher accuracy either in the low ego-depletion state or at 160 ms after hitting the ball. Additionally, there was a shorter N2 peak latency at 160 ms after hitting the ball than at the moment of hitting the ball. The peak amplitudes of N2, P3, and LNC mean amplitude were significantly lower under the low ego-depletion state than those under the high ego-depletion state (p < 0.05). The peak amplitudes of N2 and P3 were higher at the moment of hitting the ball than at 160 ms after hitting the ball. Under negative emotion, tennis-trained undergraduates exhibited faster perceptual anticipation speed, higher accuracy, and a reduced allocation of cognitive resources in a low ego-depletion state. Compared to the moment of hitting the ball, tennis-trained undergraduates exhibited better speed-accuracy, earlier attention control, and reduced allocation of attentional and cognitive resources at 160 ms after hitting the ball. The findings suggest that tennis coaches should enhance players' emotional self-regulation skills, optimize training environments, and reduce ego depletion to improve their performance.

The paper aims to investigate the difference between the effect of enoxaparin alone and enoxaparin + lower limb circulation (LLC) in the prevention of deep vein thrombosis (DVT) after intertrochanteric fracture in older people and to provide a basis for clinical anticoagulant therapy. A total of 121 patients undergoing hip fracture (HF) surgery were divided into the enoxaparin group (71 patients) and the enoxaparin + LLC group (50 patients) to compare the baseline features, changes in D-Dimer Index (DDI), and the incidence and distribution of DVT between the two groups. There was no significant difference between the baseline data of the two groups (p > 0.05). There was no significant difference between the D-Dimer Levels (DDL) 1 day after surgery (p = 0.191). But 3 days after surgery, the D-Dimer level was significantly lower in the enoxaparin + LLC group than in the enoxaparin alone group (p < 0.05). The incidence of DVT in both groups was 35.21% and 36.00% and was not statistically different (p > 0.05). No significant difference was seen in the distribution of DVT types between the two groups (p > 0.05). However, there were two popliteal vein thromboses and one femoral vein thrombosis in the enoxaparin alone group. The enoxaparin + LLC group was better at reducing the D-Dimer level than the enoxaparin group, but there was no significant difference in the incidence and distribution type of DVT. It is recommended that the proper anticoagulation regimen be selected based on the patient's risk and that the postoperative D-Dimer monitoring be supported.

Prostate cancer (PCa) is a highly heterogeneous malignancy and a leading cause of cancer-related mortality in men, with significant clinical challenges in addressing treatment resistance and disease progression. Existing preclinical models, such as two-dimensional (2D) cell lines and patient-derived xenografts (PDXs), have limitations in faithfully recapitulating the complexity of PCa, including tumor heterogeneity, androgen receptor (AR)-dependent signaling, and microenvironmental interactions. To address this gap, this study presents a robust and reproducible protocol for establishing Patient-derived organoids (PDOs) that preserve the genetic, phenotypic, and histological features of the parental tumors. This method enables the culture of organoids from both localized and advanced PCa, providing a biologically relevant model for studying disease mechanisms, drug response, and biomarker discovery. The results demonstrate the protocol's ability to generate organoids from a variety of clinical specimens, with applications in high-throughput drug screening and precision medicine. By offering a standardized workflow that addresses key technical challenges, this study highlights the importance of PDOs as versatile tools for advancing prostate cancer research and developing more effective therapeutic strategies.

Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.

Kozo washi is an archival-grade paper commonly used in the conservation of museum objects. This paper can be combined with widely used archival adhesives to form lightweight composites of natural fibers and thermoplastic resin, which can be used in fossil preparation. Unlike conventional gypsum compounds, epoxy, or metal supports, paper-resin composites are easily reversed and are made from archival materials, so they conform to modern conservation standards. We present the new "Mayborn Method," a suite of techniques for reconstructing fossil specimens while retaining suitability for radiographic study. Paper-resin composite is used to fill gaps between fragments, reinforce points of contact, and mount acrylic struts inside specimens, creating internal structural supports. Specimens prepared with these composites can withstand orientation along multiple planes for optimal imaging during X-ray and computed tomography analyses. Once a specimen is durable enough to withstand handling, it can be packed for transport with polyethylene foam and stretch wrap, which can remain in place during radiographic imaging. These techniques can be integrated throughout the fossil conservation process, from application in the field to preparation for display. These composites are strong enough to support large, heavy specimens, yet can conform to the contours of small, delicate specimens. Importantly, these techniques can stabilize "sub-fossil" material, broadening opportunities for research on these fragile specimens.

The purpose of this study is to investigate how a multidisciplinary team (MDT)-based refined nursing approach enhances clinical outcomes (including hemostatic efficacy, nutritional improvement, psychological well-being, rebleeding rates, and life quality) in elderly liver cirrhosis (LC) patients complicated with upper gastrointestinal bleeding (UGIB). Using a prospective randomized controlled trial design, 120 elderly LC + UGIB patients (2023-2024) were randomized to receive either routine care (control group, n = 60) or MDT-based refined nursing (intervention group, n = 60). MDT-refined nursing includes 3-phase interventions: acute phase (24-72 h) with half-hourly vital sign monitoring and enteral nutrition initiation within 6 h of hemostasis; recovery phase (3-7 days) incorporating pharmacist-led medication reconciliation and biweekly psychotherapy; and 1-year follow-up with telemedicine support. The main outcome measures were hemostasis time, blood transfusion volume, blood pressure control, self-care ability, nutritional status, psychological well-being, short-term adverse events, 1-year rebleeding incidence, and quality of life. The results of the study showed that the intervention group showed better outcomes in hemostasis, nutrition, psychological status, and quality of life, but survival was unaffected. However, quality-of-life measures favored the intervention group. In conclusion, MDT-based refined nursing optimizes hemostasis efficiency, nutrition, and mental health in elderly LC patients with UGIB while lowering rebleeding risk and enhancing life quality.

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly found at the interface between chromatin and the interchromatin space called euchromatin. At present, the characterization of eu- and heterochromatin is mostly based on epigenetic modifications of histone proteins along the DNA sequence, while little is known about absolute DNA densities across the cell nucleus and their functional implications. Models of the nucleus solely based on biochemical data and assumptions about the nature of chromatin as a polymer still differ fundamentally from imaging data generated by high-resolution microscopy. This indicates that there is still important structurally relevant information missing. We believe that spatial constraints might be involved in gene regulation and have therefore developed a method that allows the measurement of absolute DNA densities in mammalian cell nuclei by transforming super-resolution localization data into true-to-scale density maps by Voronoi tessellation.

This case report presents a 77-year-old male with purplish-red, blister-like lesions on the right forearm. A background of long-term corticosteroid therapy for pemphigus led to significant immunosuppression. Initial laboratory investigations, including bacterial and fungal cultures as well as histopathological analysis, yielded inconclusive results. To identify the causative pathogen, next-generation sequencing (NGS) was performed on tissue samples, which confirmed the presence of Scedosporium apiospermum, a rare opportunistic fungus. Based on this result, a targeted treatment strategy was implemented, involving multiple surgical debridements and systemic antifungal therapy with voriconazole. The therapeutic response was favorable, with complete resolution of the infection and no signs of recurrence during an 8-month follow-up period. This case highlights the clinical value of NGS in diagnosing rare and atypical infections, particularly in immunocompromised settings where conventional methods may fail. It also underscores the importance of a multidisciplinary treatment approach that integrates timely surgical intervention with appropriate antifungal therapy to achieve optimal patient outcomes.

In the field of regenerative medicine, mesenchymal stem cells (MSCs) have sparked particular attention. MSCs from the umbilical cord (UC) tissue, referred to as Wharton's jelly, have been particularly well-established in a variety of applications. In comparison to other MSC sources, Wharton's jelly MSCs (WJ-MSCs) offer a number of benefits and are isolated from UC, which is typically disposed of as medical waste. WJ-MSCs, therefore, do not raise any ethical issues similar to those pertaining to embryonic stem cells. A simple and reproducible explant technique was used to isolate WJ-MSCs from UC tissue. Within five to ten days of isolation, the WJ-MSCs emerge as adherent cells with a fibroblastic shape. After being isolated, the cells reach about 80% confluency in 14-18 days. Following that, in passage 3, the isolated WJ-MSCs was fully characterized by inducing them to differentiate along the adipogenic, osteogenic, and chondrogenic mesenchymal lineages. Additionally, immunophenotyping for negative cluster of differentiation (CD) surface markers, including CD34, CD14, and HLA-DR, as well as distinctive MSC surface markers like CD105, CD90, CD73, and CD44 were tested. Furthermore, precipitating reagent was used in a straightforward approach to isolate exosomes/small extracellular vesicles (sEVs) from the conditioned medium of those WJ-MSCs. After being isolated, exosomes/sEVs were observed under a transmission electron microscope, and particle size analysis was used to calculate their average size. In summary, WJ-MSCs provide a readily available, non-invasive, renewable supply of stem cells that may be used as factories to produce exosomes/sEVs. These WJ-MSCs and their derived exosomes/sEVs can be used in a wide array of therapeutic and downstream research applications.

Yangjing Zhongyu Decoction (YZD), a traditional Chinese herbal formula, has been suggested to improve ovarian function and fertility outcomes, but standardized clinical evidence remains limited. This study evaluated the effects of YZD in women with diminished ovarian reserve (DOR) undergoing IVF/ICSI-ET, alongside mechanistic validation in a rat model of premature ovarian failure (POF). In the clinical trial, DOR patients received either the standard GnRH antagonist protocol alone or in combination with YZD. Baseline characteristics between groups were comparable, and the YZD group showed higher numbers of retrieved oocytes and high-quality embryos, as well as improved traditional Chinese medicine syndrome scores. In parallel, POF was induced in rats, and animals treated with YZD exhibited improved estrous cyclicity, restoration of serum estradiol levels, reduction of follicle-stimulating hormone, and healthier ovarian histology. Electron microscopy revealed that YZD reduced excessive autophagosome formation and lipid accumulation in ovarian cells, suggesting regulation of the autophagy-lipid metabolism axis. Together, these findings indicate that YZD enhances ovarian reserve function, improves embryo development, and may increase pregnancy potential in women with DOR. This standardized protocol provides a reproducible framework for integrating YZD into assisted reproductive technology cycles, supporting its translational potential in clinical reproductive medicine.

Extracellular vesicles (EVs), nanosized particles approximately 20-1000 nm in size, are rapidly garnering attention for their therapeutic potential. Specifically, human breast milk-derived extracellular vesicles (HBMDEVs) have been shown to confer protection in experimental models of necrotizing enterocolitis, an intestinal disease that primarily affects premature infants, resulting in mortality as high as 50%. However, traditional extracellular vesicle isolation techniques, such as differential ultracentrifugation, size exclusion chromatography, etc., are either time-consuming or require specialized instrumentation, neither of which is practical in the clinical setting. Hence, there is a need for a simple isolation method that will allow for rapid reactive administration of HBMDEVs to patients. Proposed here is a combination of centrifugation of human breast milk to remove fat and cellular debris, coupled with rapid ultrafiltration of the remaining skim milk that produces high yield and functional extracellular vesicles. Using this technique may allow for the use of HBMDEVs as a practical therapeutic option in the clinical setting.

The transforaminal (TF) and interlaminar (IL) approaches compose the two endoscopic approaches to lumbar discectomy. There is a growing tendency to utilize the TF route for higher lumbar disc herniations and the IL approach related to anatomical factors for the caudal lumbar levels. The L5-S1 level produces a particular challenge due to anatomical barriers, including the height of the iliac crest and the orientation of the facet when confronted with a lateral or far-lateral disc herniation. Traditional approaches can lead to significant joint destabilization in the effort to resect the disc fragment    Here we present an operative video with technical nuances of the TF approach utilized to perform both a far-lateral and paracentral discectomy at L5-S1 to treat a 52-year-old male with two distinct and noncontiguous herniations with severe acute right-sided L5 and S1 radiculopathy. This would have otherwise required either two different endoscopic approaches or a more extensive open approach for successful discectomy .

Circulating tumor cells (CTCs) serve as a promising biomarker for tracking cancer metastasis, progression, and recurrence. Liquid biopsy techniques centered on CTC detection have demonstrated considerable potential due to their non-invasive nature and ability to provide real-time monitoring of tumor dynamics. However, conventional bulk CTC analyses fail to capture the intrinsic heterogeneity among CTC populations, obscuring crucial insights into tumor biology. Single-cell RNA sequencing (scRNA-seq) enables high-resolution characterization of CTC heterogeneity, offering new opportunities for precision oncology and mechanistic studies of tumor progression. Despite these advantages, the existing methodologies for single-CTC sequencing tend to suffer from inefficiencies, including low recovery rates, labor-intensive workflows, and contamination risks associated with multiple manual handling steps. To address these limitations, we present an integrated microfluidic protocol that consolidates CTC enrichment, purification, and single-cell sequencing into a unified workflow. The method employs dynamically controlled magnetic capture within a herringbone-structured chip, where vortex mixing and cumulative immunomagnetic bead binding enable robust, high-throughput CTC isolation with minimal cell damage. Subsequent purification using a leukocyte antibody-coated microfluidic chip effectively removes the non-target cells, further enhancing CTC purity through negative selection. Finally, a high-precision single-cell sequencing chip, designed based on differential flow resistance principles, facilitates efficient single-cell capture and pairing with uniquely barcoded microbeads. This novel platform overcomes the limitations of Poisson distribution-based methods, improving CTC utilization while minimizing microbead consumption and sequencing costs. Our integrated protocol significantly enhances CTC capture efficiency, purity, and single-cell sequencing throughput, making it well-suited for clinical applications and large-scale cancer research. By enabling a more precise and scalable analysis of CTC heterogeneity, this method has the potential to refine early cancer diagnosis, treatment monitoring, and mechanistic studies of metastasis, ultimately advancing the field of precision oncology.

Ultrasound Localization Microscopy (ULM) is a super-resolution imaging technique that enables in vivo visualization of the brain's microvascular architecture surpassing the diffraction limit of conventional ultrasound. By detecting and tracking intravenously injected microbubbles as they circulate through cerebral vessels, ULM produces high-resolution maps of vascular density, flow velocity, and backscattered signal amplitude at spatial scales down to 5-10 µm. This protocol presents a complete workflow for performing ULM imaging in rodents, including animal preparation, probe positioning, image acquisition, microbubble injection, and data processing, using a dedicated functional ultrasound platform. Two preparation methods are described, adapted for mice (transcranial) and rats (with cranial windows), followed by detailed instructions for probe alignment and anatomical targeting using an integrated brain atlas. During acquisition, ultrafast ultrasound sequences are synchronized with bolus injections of microbubbles to capture dynamic flow data. Subsequent reconstruction steps involve clutter filtering, image interpolation, microbubble detection, subpixel localization, and trajectory tracking. Outputs include density maps reflecting vessel occupancy, velocity maps revealing flow patterns and directionality, and amplitude maps offering additional contrast for structural interpretation. Representative results illustrate successful acquisition across full coronal planes and highlight common pitfalls such as poor injection quality, motion artifacts, and skull-induced aberration. The protocol is compatible with both cross-sectional and longitudinal studies and is particularly suited for investigating cerebrovascular alterations in models of aging, stroke, aneurysm, and neurodegenerative diseases. By combining depth penetration, high spatiotemporal resolution, and label-free vascular imaging, ULM offers a powerful tool for noninvasive brain microcirculation analysis in preclinical models.

Ischemia-reperfusion (IR) injuries are associated with several pathologies, including testicular injury. Earlier animal models of testicular IR, which involves twisting the spermatic cord to achieve ischemia, are fraught with many technical difficulties that result in the inability to reproduce the model consistently. Hence, we present a simple method for inducing testicular IR in a rat model that can easily be replicated. This method involves the separation of the gubernaculum from the testis, creation of a scrotal pouch, twisting of the testis 720° clockwise at its lower pole, and anchorage of the testis to the base of the scrotum. This model effectively constricts testicular vessels and induces testicular ischemia. After 1 h of ischemia, the anchoring stitch is removed and the testis untwisted. The cord and its accompanying vessels are inspected to ensure patency. After IR, the testis is collected and sectioned for histopathological evaluation. Testicular histology was examined for focal hemorrhagic lesions, vascular congestion, widened interstitial space, and infiltration of inflammatory cells. Furthermore, markers of oxidative stress (malondialdehyde, reduced glutathione, catalase, and superoxide dismutase), inflammation (MPO, TNF-α, IL-1β), and apoptosis (caspase 3 expression) were assayed. This method provides a simpler and easily reproducible model to study the pathophysiology of testicular IR injury. More so, this model opens a window for exploring potential therapeutic agents in the management of testicular IR injury.

Segmenting tubular structures in dense biological tissues from 3D fluorescence microscopy images is critical to study complex tissue but remains challenging due to image complexity, variability, and quality issues. Here, we introduce an open-source, user-friendly toolbox for end-to-end segmentation of tubular structures in 3D images, accessible to researchers without formal programming training. The toolbox features interactive Jupyter notebooks implementing two simple yet efficient deep learning architectures -- 3D U-Net and 3D U-Net with attention mechanisms -- for precise 3D segmentation of tubular networks. A key innovation is our simulation-based data augmentation strategy, which enhances model performance even with minimal training data (as few as one 3D image). Employing user-provided masks, the protocol generates artificial microscopy images with varying signal-to-noise ratios and simulates realistic imaging artifacts, including uneven staining, point spread function convolution, axial intensity variations, and Poisson and Gaussian noise. The protocol systematically guides users through data augmentation, model training, qualitative and quantitative evaluation on test sets, and inference on new images. We validate the toolbox by analyzing two morphologically distinct tubular networks in mouse liver tissue -- the bile canaliculi and sinusoidal networks -- demonstrating that both architectures perform well, with the attention U-Net slightly outperforming the standard U-Net when trained with augmented data. Our comprehensive toolbox, executable on local Graphics Processing Units (GPUs), high-performance computing clusters, or cloud platforms, contributes to the democratization of advanced image analysis for a broad spectrum of researchers.

In vivo confocal microscopy (IVCM) has gained significant attention for its non-invasive nature and high resolution. It enables in vivo observation and objective quantification of numerous dry eye disease (DED)-related ocular surface structures, including the cornea, conjunctiva, eyelid margin, meibomian glands, and parasites such as Demodex, at the cellular level. This capability assists ophthalmologists in identifying specific etiologies and subtypes of DED, thereby facilitating precise diagnosis, targeted treatment, and prognostic evaluation. However, standardized protocols for IVCM operation and reporting remain lacking. Proficiency in both ophthalmic knowledge and technical operation is essential to effectively utilize this device and generate high-quality reports. This article details the procedural steps and nuances of IVCM examination for DED patients. This is the first JoVE step-by-step IVCM protocol for DED. It aims to serve as a reference for clinicians, helping them avoid operational inefficiencies and consistently obtain high-quality corneal and ocular surface images to guide clinical management.