Single Technology Research Articles

Onward to Better Surgery - the Critical Need for Improved Ex Vivo Testing and Training Methods.

Guided surgery has demonstrated significant improvements in patient outcomes in some disease processes. Interest in this field has led to substantial growth in the technologies under investigation. Most likely no single technology will prove to be "best," and combinations of macro- and microscale guidance-using radiological imaging navigation, probes (activatable, perfusion, and molecular-targeted; large- and small-molecule), autofluorescence, tissue intrinsic optical properties, bioimpedance, and other characteristics-will offer patients and surgeons the greatest opportunity for high-success/low-morbidity medical interventions. Problems are arising, however, from the lack of valid testing formats; surgical training simulators suffer the same problems. Small animal models do not accurately recreate human anatomy, especially in terms of tissue volume. Large animal models are expensive and have difficulty replicating many pathological states, particularly when molecular specificity for individual cancers is required. Furthermore, the sheer number of technologies and the potential for synergistic combination leads to exponential growth of testing requirements that is unrealistic for in vivo testing. Therefore, critical need exists to expand the ex vivo/in vitro testing platforms available to investigators and, once validated, a need to increase the acceptance of these methods for funding and regulatory endpoints. Herein is a review of the available ex vivo/in vitro testing formats for guided surgery, a review of their advantages/disadvantages, and consideration for how our field may safely and more swiftly move forward through stronger adoption of these testing and validation methods.

Serum neurofilament light for detecting disease activity in individual patients in multiple sclerosis: A 48-week prospective single-center study

Background: Serum neurofilament light (sNfL) reflects neuroaxonal damage and is now used as an outcome in treatment trials of relapsing-remitting multiple sclerosis (RRMS). However, the diagnostic properties of sNfL for monitoring disease activity in individual patients warrant further investigations. Method: Patients with suspected relapse and/or contrast-enhancing lesions (CELs) were consecutively included and performed magnetic resonance imaging (MRI) of the brain at baseline and weeks 28 and 48. Serum was obtained at baseline and 2, 4, 8, 16, 24, and 48 weeks. Neurofilament light concentration was measured using Single molecule array technology. Results: We included 44 patients, 40 with RRMS and 4 with clinically isolated syndrome. The median sNfL level peaked at 2 weeks post-baseline (14.6 ng/L, interquartile range (IQR); 9.3–31.6) and reached nadir at 48 weeks (9.1 ng/L, IQR; 5.5–15.0), equivalent to the median sNfL of controls (9.1 ng/L, IQR; 7.4–12). A baseline Z-score of more than 1.1 (area under the curve; 0.78, p < 0.0001) had a sensitivity of 81% and specificity of 70% to detect disease activity. Conclusion: One out of five patients with relapse and/or CELs did not change significantly in post-baseline sNfL levels. The utility of repeated sNfL measurements to monitor disease activity is complementary rather than a substitute for clinical and MRI measures.

Simplifying stable CHO cell line generation with high probability of monoclonality by using microfluidic dispensing as an alternative to fluorescence activated cell sorting.

Single cell cloning is a critical step for cell line development (CLD) for therapeutic protein production, with proof of monoclonality being compulsorily sought in regulatory filings. Among the different single cell deposition technologies, we found that fluorescence activated cell sorting (FACS) offers high probability of monoclonality and can allow selective enrichment of the producer cells. However, FACS instruments are expensive and resource-intensive, have a large footprint, require highly skilled operators and take hours for setup, thereby complicating the cell line generation process. With the aim of finding an easy-to-use alternative to FACS, we identified a flow cytometry-based microfluidic cell dispenser, which presents a single cell sorting solution for biopharmaceutical CLD. The microfluidic cell dispenser is small, budget-friendly, easy-to-use, requires lower-cost consumables, permits flow cytometry-enabled multiparametric target cell enrichment and offers fast and gentle single cell dispensing into multiwell plates. Following comprehensive evaluation, we found that single cell deposition by the microfluidic cell dispenser resulted in >99% probability of monoclonality for production cell lines. Moreover, the clonally derived producer cell lines generated from the microfluidic cell dispenser demonstrated comparable or improved growth profiles and production capability compared to the FACS derived cell lines. Taken together, microfluidic cell dispensing can serve as a cost-effective, efficient and convenient alternative to FACS, simplifying the biopharmaceutical CLD platform with significant reductions in both scientist time and running costs.

The landscape of immune dysregulation in pediatric sepsis at a single-cell resolution

Recognizing immune dysregulation as a hallmark of sepsis pathophysiology, leukocytes have attracted major attention of investigation. While adult and pediatric sepsis are clinically distinct, their immunological delineation remains limited. Single cell technologies facilitated the characterization of immune signatures. We tackled to delineate immunological profiles of pediatric sepsis at a single-cell level by analyzing blood samples from six septic children, at both acute and recovery phases, and four healthy children. 16 single-cell transcriptomic datasets were analyzed and compared to adult sepsis dataset. We showed a unique shift in neutrophil subpopulations and functions between acute and recovery phases, along with the regulatory role of resistin. Neutrophil signatures were comparable between adult and pediatric sepsis. Innate-like CD4 T cells were predominantly and uniquely observed in acute phase of pediatric sepsis. Our study serves as a rich source of information about the phenotypic diversity and trajectory of circulating immune cells during pediatric sepsis.

Pi-seq—A customizable multichannel syringe pump for microfluidics

The advent of single cell technologies resulted in growing demand for microfluidics in the biological sciences. Commercial platforms have remained expensive, inflexible, and non-customizable black boxes. We developed an open source, multichannel, zero-backflow microfluidics device based on syringe pumps controlled by a Raspberry Pi computer. It uses both readily available and 3D-printed parts as well as a custom PCB and is easily serviceable. Moreover, it is fully customizable for various applications. Total cost is under €600. We equipped one channel with a custom Peltier-based temperature controller for precise heating or cooling and a mixer mechanism to prevent sedimentation of the cells within the syringe. Depending on the cells in the sample, heating and cooling can be useful to maintain a beneficial environment or to slow down cellular processes and cell death, respectively. Combined with microfluidics consumables and a microscope, the device is capable of integration into a high quality droplet-based single cell RNA sequencing workflow as shown here. Analysis of a mixture of human and insect cells resulted in a dataset of 17,769 single cells and demonstrates reliable operation and separation.

Abstract A087: An ovarian cancer scRNA-seq atlas to dissect tumor-host interactions underlying metastatization and chemoresistance

Abstract The high tumor heterogeneity of high grade serous ovarian cancer (HGSOC) both intra-tumoral and inter-patient is one of the main factors hampering the identification of novel therapeutic treatment to improve survival of HGSOC patients. Single cell technologies are affording new inroads for the deep molecular characterization of such tumor heterogeneity, in both primary and metastatic samples. To this aim, we built a manually curated HGSOC transcriptomics atlas (~ 1,5 million cells, 79 patients) based on publicly available datasets and developed a data integration strategy specifically applicable to very heterogenous single cell data. scRNA-seq data integration is a central methodological challenge related to single cell data analysis, particularly in the context of highly heterogenous datasets. In this context, the main source of variability is the one existing among patients. Previously developed integration methods tend to perform badly on cancer data since they mainly rely on the assumption that cell populations present in different samples are similar, which might not apply for unknown, rare or patient-specific cell populations. Here we show a novel integration approach for patient-derived cancer data. To obtain a more faithful representation of the data, we performed identification of cell populations and metacells derivation separately for every sample to identify the distinct subpopulations characterizing each patient. In addition, to robustly represent the space describing the three main cell populations of the dataset, i.e., tumoral, immune and stromal, for each of the latter we selected the space identified by the union of highly variable genes, computed separately on every dataset, to preserve the variability of the system. Next, we fed a variational autoencoder with metacells data to integrate and characterize the different subpopulations that may be similar across samples. After integration, we were able to identify new robust gene signatures that would have been masked by datasets heterogeneity. The high degree of accuracy in the manual curation of metadata harmonization in the HGSOC atlas and the robustness and scalability of the data integration pipeline allowed us to obtain an in-depth characterization of the tumor subpopulations involved in therapy response by characterizing the chemotherapy induced transcriptional features. At the same time, thanks to the atlas-associated cell types labelling strategy, this atlas represents a transformative resource for the community by enabling the investigation of the interaction between tissue microenvironment (TME) cells and tumoral cells in shaping the metastatic process. This innovative HGSOC atlas will provide the scientific community of ovarian cancer research with a very powerful tool to investigate many aspects of this disease. The robust deep learning-based framework allows to expand the atlas easily and iteratively with newly generated datasets (out of sample extension), will constitute an invaluable resource for the HGSOC scientific field tackling HGSOC pathogenetic mechanism. Citation Format: Marta R. Sallese, Pietro Lo Riso, Carlo Emanuele Villa, Giuseppe Testa. An ovarian cancer scRNA-seq atlas to dissect tumor-host interactions underlying metastatization and chemoresistance [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A087.

Exploring the metastasis-related biomarker and carcinogenic mechanism in liver cancer based on single cell technology

BackgroundHepatocellular carcinoma (HCC) is a fatal primary malignancy characterized by high invasion and migration. We aimed to explore the underlying metastasis-related mechanism supporting the development of HCC. MethodsThe dataset of single cell RNA-seq (GSE149614) were collected for cell clustering by using the Seurat R package, the FindAllMarkers function was used to find the highly expression and defined the cell cluster. The WebGestaltR package was used for the GO and KEGG function analysis of shared genes, the Gene Set Enrichment Analysis (GSVA) was performed by clusterProfiler R package, the hTFtarget database was used to identify the crucial transcription factors (TFs), the Genomics of Drug Sensitivity in Cancer (GDSC) database was used for the drug sensitivity analysis. Finally, the overexpression and trans-well assay was used for gene function analysis. ResultsWe obtained 9 cell clusters from the scRNA-seq data, including the nature killer (NK)/T cells, Myeloid cells, Hepatocytes, Epithelial cells, Endothelial cells, Plasma B cells, Smooth muscle cells, B cells, Liver bud hepatic cells. Further cell ecological analysis indicated that the Hepatocytes and Endothelial cell cluster were closely related to the cancer metastasis. Subsequently, the NDUFA4L2-Hepatocyte, GTSE1-Hepatocyte, ENTPD1-Endothelial and NDUFA4L2-Endothelial were defined as metastasis-supporting cell clusters, in which the NDUFA4L2-Hepatocyte cells was closely related to angiogenesis, while the NDUFA4L2-Endothelial was related with the inflammatory response and complement response. The overexpression and trans-well assay displayed that NDUFA4L2 exhibited clearly metastasis-promoting role in HCC progression. ConclusionWe identified and defined 4 metastasis-supporting cell clusters by using the single cell technology, the specify shared gene was observed and played crucial role in promoting cancer progression, our findings were expected to provide new insight in control cancer metastasis.

Integration of Different Storage Technologies towards Sustainable Development—A Case Study in a Greek Island

The necessity for transitioning to renewable energy sources and the intermittent nature of the natural variables lead to the integration of storage units into these projects. In this research paper, wind turbines and solar modules are combined with pumped hydro storage, batteries, and green hydrogen. Energy management strategies are described for five different scenarios of hybrid renewable energy systems, based on single or hybrid storage technologies. The motivation is driven by grid stability issues and the limited access to fresh water in the Greek islands. A RES-based desalination unit is introduced into the hybrid system for access to low-cost fresh water. The comparison of single and hybrid storage methods, the exploitation of seawater for the simultaneous fulfillment of water for domestic and agricultural purposes, and the evaluation of different energy, economic, and environmental indices are the innovative aspects of this research work. The results show that pumped hydro storage systems can cover the energy and water demand at the minimum possible price, 0.215 EUR/kWh and 1.257 EUR/m3, while hybrid storage technologies provide better results in the loss of load probability, payback period and CO2 emissions. For the pumped hydro–hydrogen hybrid storage system, these values are 21.40%, 10.87 years, and 2297 tn/year, respectively.

Analysis of single nuclear chromatin accessibility reveals unique myeloid populations in human pancreatic ductal adenocarcinoma.

A better understanding of the pancreatic ductal adenocarcinoma (PDAC) immune microenvironment is critical to developing new treatments and improving outcomes. Myeloid cells are of particular importance for PDAC progression; however, the presence of heterogenous subsets with different ontogeny and impact, along with some fluidity between them, (infiltrating monocytes vs. tissue-resident macrophages; M1 vs. M2) makes characterisation of myeloid populations challenging. Recent advances in single cell sequencing technology provide tools for characterisation of immune cell infiltrates, and open chromatin provides source and function data for myeloid cells to assist in more comprehensive characterisation. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC-Seq), a method to analyse open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumours. Frozen pancreatic tissues (benign or PDAC) were prepared for snATAC-Seq using 10× Chromium technology. Signac was used for preliminary analysis, clustering and differentially accessible chromatin region identification. The genes annotated in promoter regions were used for Gene Ontology (GO) enrichment and cell type annotation. Gene signatures were used for survival analysis with The Cancer Genome Atlas (TCGA)-pancreatic adenocarcinoma (PAAD) dataset. Myeloid cell transcription factor activities were higher in tumour than benign pancreatic samples, enabling us to further stratify tumour myeloid populations. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions for myeloid populations, including interleukin-1b signalling (recruited monocytes) and intracellular protein transport (dendritic cells). The identified gene signature for dendritic cells influenced survival (hazard ratio=.63, p=.03) in the TCGA-PAAD dataset, which was unique to PDAC. These data suggest snATAC-Seq as a method for analysis of frozen human pancreatic tissues to distinguish myeloid populations. An improved understanding of myeloid cell heterogeneity and function is important for developing new treatment targets in PDAC.

Cross-sectional analysis of use of real-world data in single technology appraisals of oncological medicine by the National Institute for Health and Care Excellence in 2011–2021

ObjectivesThis study aims to identify how real-world data (RWD) have been used in single technology appraisals (STAs) of cancer drugs by the National Institute for Health and Care Excellence (NICE).DesignCross-sectional...

The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies.

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes.

Digital skills within the Public Sector: A missing link to achieve the Sustainable Development Goals (SDGs)

Academic literature has mostly discussed digital skills concerning end-users’ capability to access and use single technologies. Against this background, the paper sheds light on a missing element: the digital literacy of those who frame ICT-mediated policies that pursue sustainable development. The paper offers a novel conceptualization of digital skills as the capabilities to understand the socio-technical assemblages that emerge in social contexts after the adoption of ICT-mediated policies. Exploring the case study of the United Kingdom’s Government Digital Service, the paper argues that empowering public administrators and civil servants with these digital skills is paramount to design, implement, and manage ICT-mediated policies that aim to achieve Sustainable Development Goals.

Label-Free Optical Imaging of Nanoscale Single Entities.

The advancement of optical microscopy technologies has achieved imaging of nanoscale objects, including nanomaterials, virions, organelles, and biological molecules, at the single entity level. Recently developed plasmonic and scattering based optical microscopy technologies have enabled label-free imaging of single entities with high spatial and temporal resolutions. These label-free methods eliminate the complexity of sample labeling and minimize the perturbation of the analyte native state. Additionally, these imaging-based methods can noninvasively probe the dynamics and functions of single entities with sufficient throughput for heterogeneity analysis. This perspective will review label-free single entity imaging technologies and discuss their principles, applications, and key challenges.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

Non-Invasive Survey Techniques to Study Nuragic Archaeological Sites: The Nanni Arrù Case Study (Sardinia, Italy)

The Italian territory of Sardinia Island has an enormous cultural and identity heritage from the Pre-Nuragic and Nuragic periods, with archaeological evidence of more than 7000 sites. However, many other undiscovered remnants of these ancient times are believed to be present. In this context, it can be helpful to analyze data from different types of sensors on a single information technology platform, to better identify and perimeter hidden archaeological structures. The main objective of the study is to define a methodology that through the processing, analysis, and comparison of data obtained using different non-invasive survey techniques could help to identify and document archaeological sites not yet or only partially investigated. The non-invasive techniques include satellite, unmanned aerial vehicle, and geophysical surveys that have been applied at the nuraghe Nanni Arrù, one of the most important finds in recent times. The complexity of this ancient megalithic edifice and its surroundings represents an ideal use case. The surveys showed some anomalies in the areas south–east and north–east of the excavated portion of the Nanni Arrù site. The comparison between data obtained with the different survey techniques used in the study suggests that in areas where anomalies have been confirmed by multiple data types, buried structures may be present. To confirm this hypothesis, further studies are believed necessary, for example, additional geophysical surveys in the excavated part of the site.

Changing streetlighting schemes and the ecological availability of darkness.

Artificial light at night (ALAN), from streetlights and other sources, has a wide variety of impacts on the natural environment. A significant challenge remains, however, to predict at intermediate spatial extents (e.g. across a city) the ALAN that organisms experience under different lighting regimes. Here we use Monte Carlo radiative Transfer to model the three-dimensional lighting environment at, and just above, ground level, on the spatial scales at which animals and humans experience it. We show how this technique can be used to model a suite of both real and hypothetical lighting environments, mimicking the transition of public infrastructure between different lighting technologies. We then demonstrate how the behaviour of animals experiencing these simulated lighting environments can be emulated to probe the availability of darkness, and dark corridors, within them. Our simulations show that no single lighting technology provides an unmitigated alleviation of negative impacts within urban environments, and that holistic treatments of entire lighting environments should be employed when understanding how animals use and traverse them.

Farmers’ adoption of multiple climate-smart agricultural technologies in Ghana: determinants and impacts on maize yields and net farm income

This study investigates the factors affecting maize farmers’ decisions to adopt climate-smart agricultural (CSA) technologies and estimates the impacts of CSA technology adoption on maize yields and net farm income. Unlike most previous studies that analyze a single technology, we consider different combinations of three CSA technologies (zero tillage, row planting, and drought-resistant seed). A multinomial endogenous switching regression model addresses selection bias issues arising from observed and unobserved factors and analyses data collected from 3197 smallholder farmers in three Ghana regions (Brong-Ahafo, Northern, and Ashanti). The findings show that smallholder farmers’ decisions to adopt multiple CSA technologies are influenced by farmer-based organization membership, education, resource constraints such as lack of land, access to markets, and production shocks such as perceived pest and disease stress and drought. We also find that adopting all three CSA technologies together has the largest impact on maize yields, while adopting row planting and zero tillage as a combination has the largest impact on net farm income. Governments should collaborate with farmer-based groups and extension officers to improve farmers’ awareness and understanding of the benefits associated with CSA technologies and help them adopt multiple technologies that generate higher benefits.

Non-invasive efficacy assessment of pulsed dye laser and photodynamic therapy for port-wine stain.

Port wine stain (PWS) is a congenital vascular malformation that commonly occurs on the face and neck. Currently, the main treatments for port wine stain are pulsed dye laser (PDL) and photodynamic therapy (PDT). However, the efficacy evaluation of PWS mostly relies on the subjective judgement of clinicians, and it is difficult to accurately respond to many small changes after treatment. Therefore, some non-invasive and efficient efficacy assessment methods are also needed. With the continuous development of technology, there are currently many visualisation instruments to evaluate PWS, including dermoscopy, VISIA-CR™ system, reflectance confocal microscopy (RCM), high-frequency ultrasound (HFUS), optical coherence tomography (OCT), Photoacoustic imaging (PAI), laser speckle imaging (LSI) and laser Doppler imaging (LDI). Among them, there are simple and low-cost technologies such as dermoscopy and the VISIA-CR™ system, but they may not be able to observe the deeper structures of PWS. At this time, combining techniques such as HFUS and OCT to increase penetration depth is crucial to evaluate PWS. In the future, the combination of these different technologies could help overcome the limitations of a single technology. This article provides a systematic overview of non-invasive methods for evaluating treatment efficacy in port wine stains and summarises their advantages and disadvantages.

Correlation of self-reported racial background to euploidy status and live birth rates in assisted reproductive technology cycles.

To determine whether the embryonic euploidy rate and live birth outcomes following single, euploid embryo transfer (SEET) differ among women of self-reported racial and ethnic backgrounds. This retrospective cohort study included all infertile patients of different self-reported racial backgrounds who underwent In vitro fertilization (IVF) with preimplantation genetic testing for aneuploidy (PGT-A) and an autologous single euploid embryo transfer (SEET) from December 2015 to December 2019 at a single private and academic assisted reproduction technology center. Primary outcome measures included ploidy rates among different racial groups. Secondary outcomes included clinical pregnancy, clinical pregnancy loss, and live birth rates. Five thousand five hundred sixty-two patients who underwent an IVF cycle with ICSI-PGT-A were included. A total of 24,491 blastocysts were analyzed. White participants had on average more euploid embryos and higher euploidy rates when compared to their counterparts (p ≤ 0.0001). However, after controlling for confounding factors, there was no association between race and the odds of having ahigher euploidy rate (aOR 1.31; 95% CI 0.63-2.17, p = 0.42). A total of 4949 patients underwent SEET. Pregnancy outcomes did not differ among patients of varying self-reported races. Euploidy rates and pregnancy outcomes were comparable among patients of different racial backgrounds who underwent a SEET.

Single Cell Sequencing Technology and Its Application in Alzheimer's Disease.

Alzheimer's disease (AD) involves degeneration of cells in the brain. Due to insidious onset and slow progression, AD is often not diagnosed until it gets progressed to a more severe stage. The diagnosis and treatment of AD has been a challenge. In recent years, high-throughput sequencing technologies have exhibited advantages in exploring the pathogenesis of diseases. However, the types of cells of the central nervous system are complex and traditional bulk sequencing cannot reflect their heterogeneity. Single-cell sequencing technology enables study at the individual cell level and has an irreplaceable advantage in the study of complex diseases. In recent years, this field has expanded rapidly and several types of single-cell sequencing technologies have emerged, including transcriptomics, epigenomics, genomics and proteomics. This review article provides an overview of these single-cell sequencing technologies and their application in AD.