About

Daniel T. Chiu is the A. Bruce Montgomery Professor of Chemistry and the Endowed Professor in Analytical Chemistry at the University of Washington. His research focuses on analytical chemistry, with particular emphasis on developing innovative methods and techniques for chemical analysis. As a distinguished faculty member, he contributes to advancing the understanding and application of analytical methods in chemistry.

Research topics

• Biochemistry
• Cell biology
• Cancer research
• Chemistry
• Photochemistry
• Materials science
• Genetics
• Biology
• Organic chemistry

Selected publications

• Measuring the Degree of Labeling of Antibody-Dye Conjugates with a Single-Molecule-Sensitive Digital Flow Cytometer

  Analytical Chemistry · 2026-03-03

  articleOpen accessSenior authorCorresponding

  Antibody-dye conjugates are an essential tool in biomedical research and are widely used in flow cytometry, immunofluorescence imaging, and ELISAs. The degree of labeling (DOL), often referred to as the F/P (fluorophore-to-protein) ratio, which is the number of dyes per antibody, is a fundamental characteristic of antibody-dye conjugates that impacts conjugate performance (emission intensity, affinity, solubility/aggregation) and the accuracy and reproducibility of results. Knowing the DOL of antibody-dye conjugates is essential for accurately quantifying target proteins and/or antigens in quantitative flow cytometry and quantitative immunofluorescence. In CAR-T cell therapy, the expression level of the chimeric antigen receptor (CAR) on T cells must be finely tuned to optimize treatment efficacy but cannot be calculated without the DOL of the anti-CAR antibody-dye conjugate. However, DOL cannot be easily measured (e.g., via absorption spectroscopy) for many widely used conjugates that contain protein-based or semiconducting polymer/nanoparticle dyes due to overlap in absorption cross-section between antibody and dye. Bulk analysis also cannot reveal the distribution of DOL values, which can impact conjugate performance and biomarker quantitation. Furthermore, quenching between dyes can reduce the conjugate emission. Herein, we demonstrate the use of a single-molecule-sensitive digital flow cytometer (dFC) with 100% single-fluorophore detection efficiency to measure the fluorescence emission distributions of conjugates and free dyes and perform deconvolution to obtain effective DOL values (the apparent number of dyes per conjugate based on fluorescence emission, useful for converting fluorescence intensity to antigens/cell) for antibodies conjugated with small-molecule, protein-based, and semiconducting polymer/nanoparticle dyes.

  PublisherOA PDFDOI

• Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the <scp>CYTO</scp> 2025 Conference Workshops

  Cytometry Part A · 2025-12-30

  articleOpen access

  Organized by the International Society for Advancement of Cytometry (ISAC), the CYTO conference is one of the most important events for everyone interested in cytometry and quantitative single-cell analysis. It is an annual gathering of cytometry experts and novices in the field, all with a passion for cytometry. This meeting is an opportunity to learn exciting, cutting-edge research directly from the source by attending the Frontiers and State-of-the-Art lectures, research and technology sessions, and Scientific Tutorials. CYTO conferences are also the premier live forum for discussing current and emerging challenges in cytometry and associated sciences through an interactive workshop format. Workshops attract considerable attention within and outside the cytometry community as they address where we stand in specific fields of interest to cytometrists, how current issues with the evolving technologies within the field can be addressed and solved, and what future areas for development exist. Due to the limited time frame allocated to this format, however, workshops run in parallel, preventing attendees from participating in all sessions of interest. Moreover, those unable to attend the CYTO conference miss the content entirely. Therefore, we continue the tradition of summarizing all workshops to disseminate the information provided and to capture the state-of-the-art view of the community. The primary purpose of creating such a combined summary is to preserve the record of live discussions and main conclusions, propose potential guidelines, and make them available to the broader public. The publication of workshop summaries and their availability to a global audience not only captures live discussions but also undoubtedly seeds new initiatives to support emerging topics or trends. This “Cyt-Geist,” a play on the German Zeitgeist, meaning “spirit of the age”, aims to document the defining spirit and collective consciousness of the cytometry field at this moment in time. Just as Zeitgeist captures the prevailing ideas and beliefs of an era, Cyt-Geist captures the current challenges, innovations, and forward momentum that characterize our community. The joint effort begun in 2018 and continued in 2019 by Kamila Czechowska proved to be a valuable reference [1, 2]. Building on that spirit, we present summaries from CYTO 2025, held in Denver, Colorado, from May 31 to June 4. This manuscript serves as a summary report of 15 workshops held at CYTO 2025. We present, in concise form, the current and future challenges in cytometry identified by workshop organizers and participants. The manuscript is organized into three thematic sections: Building the Cytometry Infrastructure of the Future (Standardization, Sharing, and Sustainability in Cytometric Practice); Applied Innovation Across Modalities (Expanding Possibilities: From Fluorescence to Imaging and Automated Annotation); and the People Behind the Panels (Workflows, Workspaces, and the Human Side of Cytometry). Each section addresses critical aspects of modern cytometry practice, from foundational infrastructure and technical innovation to professional development and operational sustainability. We intend to serve with this joint workshop report the global community involved in single-cell analysis and cytometry. Workshops 2, 3, 7, 8, and 13 addressed the foundational frameworks needed to ensure success for the cytometry community in the years ahead. The themes ranged from pre-analytical considerations in sample handling to standardization of instruments and file formats, and from data stewardship to the future of public repositories. Together, these discussions underscored that sustainable progress in cytometry depends not only on innovation but also on building robust infrastructure, shared standards, and reliable practices. WS02 (Pre-analytical variables) examined the numerous factors that can affect the quality of peripheral blood mononuclear cells (PBMCs) and the interpretation of downstream assays, emphasizing the importance of defining the context of use (COU) and minimizing variability. WS08 (Spectral Standardization) highlighted the need for community-driven best practices to account for differences in platforms, reagents, and data analysis methods, including nomenclature and unmixing. WS13 (FCS 4.0) provided historical context for FCS file formats and outlined the community's priorities and timeline for modernizing the standard to support spectral data, interoperability, and high-dimensional analysis. WS03 (FlowRepository) focused on the sustainability and governance of this community resource, emphasizing the need for accessibility, clear licensing, and forward-looking technical development. WS07 (Data Management and Sharing) brought the perspective of Shared Resource Laboratories (SRLs), advocating for broad adoption of the FAIR data principles to support reproducibility, accessibility, and long-term scientific value. Taken together, the outcomes of these workshops point to a central conclusion: the future of cytometry depends on shared responsibility for infrastructure. By harmonizing practices, investing in sustainable platforms, and fostering a culture of open data and reproducibility, the community can ensure that cytometry remains a cornerstone of biomedical discovery. The establishment of task forces, working groups, and continued discussions through community platforms demonstrates the commitment to translating workshop insights into actionable progress. Nicolas Loof, Meredith Weglarz, Jessica Back, Benjamin Daniel, Cheryl Kim, and William Murphy Data management encompasses all disciplines related to handling data as a valuable resource, including annotation, storage, retrieval, security, and organization. A 2014 study [3] demonstrated that the odds of obtaining underlying research data dropped 17% annually after the second year of publication, emphasizing the critical need for effective data stewardship. In cytometry, rapid growth in file sizes from spectral and imaging technologies makes this challenge particularly urgent. Historically, data retention was the responsibility of individual researchers, but SRLs are increasingly accountable for data stewardship. Recent developments in spectral and imaging cytometry technologies have led to significant increases in file sizes, making data storage more challenging than ever for both users and SRLs. These shifts bring challenges around cost, scalability, and technical implementation. For many users and SRLs, data management approaches have become increasingly relevant, as is that for use file of with and are is in data management ideas and in and SRLs the including considerations for annotation, accessibility, and A community on current practices The workshop a summary of from and SRLs, and open with participants. The workshop with a summary of the that the the community. address these challenges, the workshop the adoption of the and data These principles a to data stewardship that reproducibility, and the long-term of The workshop highlighted a of that support effective data of and of shared their data management These that is and can data stewardship identified with file and remains use to is but to the of but are and with data management is a for modern not a practices can data into a cornerstone of and By the standards, and fostering a culture of data can ensure that their data remains a data management reproducibility, and discovery. It data from a of research into a cornerstone of scientific progress. The are and by their or and report of interest. and Jessica This workshop the current and future development of a public data for cytometry in cytometry, and data governance addressed the challenges of and the and of to the for robust building on governance and and future technical and issues to limited storage, and and are The community management and and The workshop attendees through interactive and A live was to on the need for and in the community at both the and of the the of the from to historical context and on current of the workshop was in into that examined the potential for or the of and the of the the to summaries of topics and on identified three central accessibility, licensing, and These are by and with limited limited and data storage, as as for and sustainability topics of including or to an also and potential with such as or with the of and Data and also as factors with emphasizing the importance of open for the of data discussions licensing, particularly to use a open that and with or a more that but not with cytometry to data Future a of the to support and long-term through infrastructure The need to address challenges to data was also It was that the with and reliable cytometry data that can be The current not and this to be addressed remains a community The workshop highlighted community support for as a for cytometry emphasizing importance in cytometry data also to the current in and infrastructure that on a the progress A task was and a was to from with a on long-term sustainability and reliable data The task primary is the development of to or the current and infrastructure. of 2025, this new is in an with on to support long-term In parallel, the task is working to a of repositories. These their data and to the they also public and for or support interested in a that the of public data and or interested in participating in the central task or creating a are to for more The are and by their or and report of interest. and The Cytometry file critical for data handling and standardization in cytometry as FCS by the International Society for Advancement of Cytometry for interoperability, data and analysis platforms, and the in FCS data and in FCS data standard and the with The FCS addressed community sample and The FCS in and in after community for and as as support for data for and this for adoption by and By the Data also FCS to support spectral and this not as and is The Data is future to a or for more data storage with modern high-dimensional This critical the FCS workshop at CYTO 2025. The workshop to the FCS timeline and community on to make or a issues standards, spectral data file and adoption by both and was to CYTO Cytometry Cytometry The identified challenges with the current FCS and FCS and The workshop the FCS timeline and The timeline for FCS for to the for Data of the for CYTO for and timeline and for publication in Cytometry attendees in live on spectral and file on these with summarizing such as a of or and imaging cytometry of for FCS as they development they be as a file discussions focused on a of priorities for an FCS file at CYTO into was on a of to the file that support as cytometry as and to the use of FCS by those data analysis for and information to support reproducibility, a for information and ensure that captures analysis than analysis outcomes to The FCS standard capture all data and needed to from data, including the spectral of the and of the of the spectral was the need for a to and FCS was on and data be within the file or on the importance of and data within FCS to of the file to be into This become increasingly valuable as file sizes continue to The also the potential of or support for or to information the of FCS an to new data and in that are both and to analysis The and workshop critical areas for in the FCS The community's on spectral data and data handling considerations the for a FCS these priorities is for data reproducibility, data and the evolving of cytometry We to including and for valuable on their with the FCS standard as as all workshop for their support of the The are and by their or and report of interest. and the the of spectral on the and they are in users are in this however, is a of more cytometry bring to the and are to have in are how to use can to in data analysis. Each also a can become instruments and and This workshop to an of the current cytometry and to the community standardization in spectral cytometry. the was to the best to The workshop specific challenges by the of spectral instruments in the field, the of data these platforms and to instruments through audience was to and from the community the workshop was to groups, the than organizers to the as a and of the audience the to their A was also the audience to The to a best practices and bring forward the for an task on spectral cytometry community be for The are and by their or and report of interest. and This workshop examined the of pre-analytical emphasizing the importance of defining and for For and assays, the of is a critical that one of the primary issues the standardization and of is that many approaches exist. defining the and are important for the development of and single-cell that use In of these the workshop was to a the attendees to the and future for in available highlighted in a CYTO on current practices for The provided on the numerous factors that quality and the these factors have on the downstream was also provided on that including and with best practices for within the focused on a on current best practices for for quality and potential to the quality of the A was shared on the and with the of the and Society of the Cytometry the the of and the Society to information on in current for to capture the challenges and priorities with the community the content of the on current best practices. the topics for continued and the audience to them for the workshop and a was in downstream and interpretation of sample sample quality the from blood and through storage, and data from as the and primary workshop within of blood on with this considerable and challenges for many the a with for This the for future to available and make associated with and blood many to a The of a for is however, are as many the for to quality and associated In a or central the but this of the workshop audience with and these into account their assays, to within an The of the workshop was the of and how to with more for these as as quality and the the workshop for future and are of The audience that be more for the specific use of the the is data and can be such as the need for quantitative This on to a or in how to best and the of from time and in and be for a study at an time point for all topics by the audience in to the and including for data of quality and approaches to the for building future best practices and for The audience as a to the of For by at within the be to the data with the for In attendees that from more that the of on and that current and around sample handling and critical to The scientific community from in the such as the of time from blood to This information more in in for data and a Cytometry highlighted as a The need for of and with the outlined in the was to our of how to attendees are that can their of interest to the from at the of in our of to The use of available to this was challenges, including a of as as associated for and need to be the potential a was the of to with from the audience of implementation. with the of this become increasingly more and time. In to or on a of at the the of with a or on a of from be as a to sample was also This be to and an to on of the workshop addressed a to that in where are the of and through and the of approaches that be more such as of these by a in was Future by interested in the working that attendees of the conference and the broader community is and can be through an account to the the was available to attendees and after the It can be through the The are and by their or and report of interest. The workshops in this section examined how cytometry is with that and new These sessions a shared to emerging technologies in that reproducibility, and highlighted the need for and frameworks in cytometry, emphasizing the of and addressed the and for on and and how quantitative approaches can and demonstrated how and can address in and as a source of than of and to bring into A clear of community-driven to approaches or to a was in all the workshops in this is to these as we the second of this with a global that an on by the and scientific community to to the of the but community-driven in cytometry continue to this field into a Together, these workshops a the cytometry community is not only but also what cytometry can with and and Cytometric analysis challenges in from to and These challenges and data to in data analysis and to The the workshop at CYTO was at the broader cytometry community involved in research The of that workshop was to the and of working with and and imaging cytometry. The of the CYTO workshop highlighted a need for sample and in and cytometry on this the to a research with cytometry. This was by the involved in handling and the significant present current practices. In a of and but with the of working with sample or and the of specific challenges cytometry issues such as the of and sample for The workshop to bring experts in the field to a of and to that be shared with the community and within cytometry SRLs and The with a summary of outcomes from the CYTO workshop by current challenges in cytometry. was live and The workshop was to open to their and The audience that of the attendees involved in cytometry, through their in an or as of their research The of the and highlighted main of and is limited and in cytometry within the broader community and are as are and is to all however, the sample from specific to this is a in most cytometry and the of be be the sample is and the of cells of interest is are not and can to in the of in the and their on sample quality are also was the on how and and The of sample and sizes for from a for and Data issues and for more of The second of the workshop addressed how emerging technologies the cytometry that and imaging cytometry new for sample The workshop with the of a working to development and standardization for the of and within in the cytometry community are to interest in the support an and to for and the Cytometry was as a forward The of sciences is in to global challenges, including to and cytometry technologies and however, the of and broader adoption and This workshop underscored the need for practices and be critical for technologies and this the an meeting of the working to and areas for of this a of and In the the of this serve as a for future in such as and cytometry. We to everyone the workshops at CYTO and 2025. We for professional to the CYTO for and The provided by with the and groups, was The are and by their and report of interest. and the rapid of imaging cytometry, is a need for around and in cytometry. of and insights This workshop addressed these emerging by in a that the and of cytometry. The insights from a where to their and of the of cytometry. and the to on and that can be and by The also in imaging their and operational In they examined the of imaging and cytometry, or in In the to to a collective for defining cytometry, standard and the that this the community was through a as both a and a This from a of and It not only technical and with cytometry but also and future By the in of the organizers that a to on their practices and the broader themes of the the a to and The with a of the and their for the is This their field and the the of available was also to the in current This was by a of the highlighted challenges, and of cytometry within the community. a live was attendees to to and their This was to an interactive and for open and collective fostering both and critical of the A was the potential for future to the evolving of cytometry and the that this continued was through A of interest in and the was the the for through future working groups, or development and insights from the are to both and in the The workshop focused on the emerging and of cytometry, particularly in from imaging and cytometry. by both the and in on challenges and priorities in the From both the and live issues and challenges how to cytometry to as a of cytometry, as more with This and development. of an was in how the as a of focused on the or This of and in the cytometry instruments the or high-dimensional data the source of the is on or be the need for on a of such as and to analysis and of on the need to both the data and the to and This support reproducibility, interoperability, and development. and technical attendees highlighted the and technical of cytometry platforms, with a of These factors broader adoption and effective use of the These are with broader discussions in the the need for and community-driven frameworks in the field of imaging and cytometry A of critical was live and and and information The workshop clear that cytometry is at a critical in but standards, and on and a working of identified the need for a that cytometry from both cytometry and This from and standardization was broad support for a of that be and in cytometry This guidelines, standards, and standardization The community a to cytometry data formats and in cytometry FCS This to the of a working in with or development of The a interest in workshop to and propose best practices for and cytometry important of this effort be to with and to ensure that are in future and task was the interest was in a community of that continue standards, and use to the value. and Future also and that support new users in and cytometry The are and by their and report of interest. and and are significant challenges in modern and numerous at both the community and are focused on in these from numerous and to the and of quantitative cytometry the years have one of the most and platforms, the of in and The workshop was a joint effort of involved in the of quantitative cytometry in and A of this of for the of an and and the of to be involved in The workshop was an of the that to current practices for to quantitative cytometry and issues and for The and The workshop a of attendees and with an of research and practices. attendees of shared users and as by a of most attendees of as a practice, a such data to characterize and to users to report their in quantitative with this as a The of the focused on the and of current best practices for and and provided a of quantitative cytometry developments the years and how these developments have to instruments and the they an of and their and their use to the of spectral and in of of and reference The of the to data quantitative data analysis and highlighted the and as as the for in spectral cytometry. of and provided a summary of from the Cytometry a effort of and involved in and research and as as of cytometry reagents, and The of these and practices a of working are and to spectral and and these to report the of and in quantitative and on that and of in from on more than instruments from These by audience and interactive standardization to of data instruments of to and more robust interpretation and of cytometry data, with and capture and the of instruments and to support quantitative cytometry. The second of the workshop focused on the scientific of quantitative cytometry. of cytometry and individual including and the importance of the of have a to the of the challenges of on individual to these and the differences approaches and approaches with an of to to instruments and to characterize at the in and be for quality to and in The workshop with a for to become of the Cytometry and by participating in an This to for at standard and to report a to in these be the with to be at a future CYTO quality capture and the cells as spectral and was significant of interest in the topics in this workshop with WS13 (FCS and Future where effective for are needed that and to support quantitative cytometry, and a where was as an of In WS08 of and in on and areas where and these potential topics for future The establishment of the Cytometry and a significant broader adoption of quantitative practices. Future for into spectral cytometry, and with related standardization workshop The are and by their or and report of interest. Kamila and The The workshop with a and but at This standardization For cells and cells these the of that of workshop in a in and as address these challenges, the a reference This a of and The not only as a but also as an for interoperability, to their data This effort the FAIR data principles in WS07 (Data Management the importance of reproducibility, accessibility, and long-term scientific the cytometry community. this a quantitative was that how are approaches such as or The new these with summaries on and These and within For both and of a new of that and the for specific These the including This by all This that standard are and from not these they use the quantitative scientific the of The technical of the workshop for data particularly forward-looking innovation on The and These and making them for the workshop The also the of a where a reference serves as the from this reference are to through making this particularly valuable for or where can a of was as the The and with The frameworks from and have demonstrated success in new data to reference These a for cytometry, that combined with and platforms also for to from data their these in The particularly through the development of a reference and the needed to into a frameworks remains the standard for

  PublisherOA PDFDOI

• Characterization of a Single-Molecule Sensitive Digital Flow Cytometer for Amplification-Free Digital Assays

  ACS Nano · 2025-06-17 · 7 citations

  articleOpen accessSenior authorCorresponding

  Digital assays such as digital PCR for nucleic acids and digital ELISA for proteins provide absolute quantitation and greater accuracy, sensitivity, and reproducibility than their analogue counterparts (real-time PCR and standard ELISA), but current digital assays involve amplification (e.g., DNA amplification in digital PCR and signal amplification in digital ELISA), which makes high multiplexing difficult, often requires complex and expensive sample compartmentalization, and adds reaction steps. We have developed a single-molecule sensitive flow cytometer, which we termed a digital flow cytometer (dFC). dFC optimizes the sensitivity and efficiency of single-molecule detection by using smaller, planar microfluidic channels, a smaller probe volume, and a shorter working distance/higher numerical aperture objective than used in current commercial high-sensitivity flow cytometers, allowing digital assays via direct single-molecule counting. This paper describes our characterization of the analytical performance of this system when detecting antibody-dye conjugates and demonstrates absolute concentration measurements of commercial antibody-dye conjugates. The dFC exhibited a single-molecule detection efficiency with which over 98% for antibodies conjugated with 18 different small-molecule, phycobiliprotein, and semiconducting polymer dyes were separated from noise, a low false-positive rate, a stable baseline signal, and accurate concentration measurements with a dynamic range spanning 4 orders of magnitude. This system can be used for authenticating antibody-dye conjugates used in flow cytometry and tissue imaging studies and in the development of multiplexed, amplification-free digital assays for nucleic acids and proteins.

  PublisherOA PDFDOI

• QOL-17. ASCENT: A population-specific ACT-based psychosocial intervention for patients with newly diagnosed malignant primary CNS tumors

  Neuro-Oncology · 2025-11-01

  articleOpen access

  Abstract BACKGROUND Patients with newly diagnosed malignant primary central nervous system (CNS) tumors experience significant distress related to their diagnosis and poor prognosis. Our prior qualitative work demonstrated that these patients often cope with distress through cognitive, emotional, and behavioral avoidance, which may hinder values-based living and portend worse psychological outcomes. Using principles of Acceptance and Commitment Therapy (ACT), we developed a population-specific intervention (“ASCENT”) aimed to reduce distress, improve coping, and promote values-driven behaviors. METHODS Building on our prior qualitative work and using ACT as a framework, our multidisciplinary team developed preliminary intervention modules targeting the areas of greatest patient distress and need. We then refined the proposed intervention content using input from an advisory council of patient and caregiver stakeholders. We additionally elicited expert input from neuropsychology and speech-language pathology colleagues to ensure accessibility for patients facing cognitive and/or communication challenges, which often affect patients with CNS tumors. RESULTS ASCENT is a six-session, psychosocial intervention consisting of weekly, one-on-one, 45–60-minute meetings with a trained clinician. Sessions build on core ACT principles, with key components including: defining the patient’s values, identifying obstacles to living by those values, practicing acceptance, mindfulness training, problem-solving and communication skills, and goal setting. Participants receive a manual containing session didactics and activities, with content and design features that accommodate patients with cognitive/communication limitations. We will refine the intervention based on stakeholder feedback and an upcoming open pilot study, prior to a randomized controlled trial to evaluate intervention feasibility and acceptability. CONCLUSION ASCENT represents a novel, population-specific approach using the tenets of ACT to address distress in patients with malignant primary CNS tumors, specifically tailored to facilitate participation of patients with cognitive and/or communication deficits. Findings from preliminary studies will inform future evaluation of ASCENT’s efficacy in improving psychological and behavioral outcomes.

  PublisherOA PDFDOI

• Extracellular Vesicles for Clinical Diagnostics: From Bulk Measurements to Single-Vesicle Analysis

  ACS Nano · 2025-07-28 · 51 citations

  reviewOpen access

  Extracellular vesicles (EVs) play a crucial role in intercellular communication, signaling pathways, and disease pathogenesis by transporting biomolecules such as DNA, RNA, proteins, and lipids derived from their cells of origin, and they have demonstrated substantial potential in clinical applications. Their clinical significance underscores the need for sensitive methods to fully harness their diagnostic potential. In this comprehensive review, we explore EV heterogeneity related to biogenesis, structure, content, origin, sample type, and function roles; the use of EVs as disease biomarkers; and the evolving landscape of EV measurement for clinical diagnostics, highlighting the progression from bulk measurement to single vesicle analysis. This review covers emerging technologies such as single-particle tracking microscopy, single-vesicle RNA sequencing, and various nanopore-, nanoplasmonic-, immuno-digital droplet-, microfluidic-, and nanomaterial-based techniques. Unlike traditional bulk analysis methods, these methods contribute uniquely to EV characterization. Techniques like droplet-based single EV-counting enzyme-linked immunosorbent assays (ELISA), proximity-dependent barcoding assays, and surface-enhanced Raman spectroscopy further enhance our ability to precisely identify biomarkers, detect diseases earlier, and significantly improve clinical outcomes. These innovations provide access to intricate molecular details that expand our understanding of EV composition, with profound diagnostic implications. This review also examines key research challenges in the field, including the complexities of sample analysis, technique sensitivity and specificity, the level of detail provided by analytical methods, and practical applications, and we identify directions for future research. This review underscores the value of advanced EV analysis methods, which contribute to deep insights into EV-mediated pathological diversity and enhanced clinical diagnostics.

  PublisherDOI

• High-Throughput Analysis of Single Extracellular Vesicles

  2025-01-01

  book-chapterSenior author
  PublisherDOI

• Heterogeneity of Extracellular Vesicles and Non‐Vesicular Nanoparticles in Glioblastoma

  Journal of Extracellular Vesicles · 2025-10-01 · 3 citations

  articleOpen access

  It is increasingly clear that intercellular communication is largely mediated by lipid-bilayer, membrane-bound extracellular vesicles (EVs) and amembranous, non-vesicular extracellular particles (NVEPs), including exomeres and the recently identified supermeres. To elucidate the cargo and functional roles of these carriers, we performed a comprehensive analysis of their lipid, protein and RNA content in the context of colorectal cancer and glioblastoma (GBM). Our results demonstrate that EVs exhibit distinct density profiles correlated with specific biomolecular signatures. Moreover, EVs and NVEPs display notable differences in their protein and RNA composition, which confer distinct functional attributes. Supermeres are notably enriched in components involved in extracellular matrix remodeling and possess the ability to cross the blood-brain barrier, a process dependent on their intact structure and RNA content. Once in the central nervous system (CNS), they preferentially engage with microglia and suppress TGFβ1 expression, suggesting a role in modulating microglial immune activity. Furthermore, systemically administered exogenous supermeres selectively accumulate in GBM tumors in vivo. Together, these findings highlight supermeres as a promising vehicle for delivering therapeutics to the CNS and brain tumors.

  PublisherOA PDFDOI

• Humanitarian engineering solutions: medical oncology

  2024-06-01

  book-chapterSenior author
  PublisherDOI

• A perspective from the National Eye Institute Extracellular Vesicle Workshop: Gaps, needs, and opportunities for studies of extracellular vesicles in vision research

  Journal of Extracellular Vesicles · 2024-12-01 · 9 citations

  reviewOpen access

  With an evolving understanding and new discoveries in extracellular vesicle (EV) biology and their implications in health and disease, the significant diagnostic and therapeutic potential of EVs for vision research has gained recognition. In 2021, the National Eye Institute (NEI) unveiled its Strategic Plan titled 'Vision for the Future (2021-2025),' which listed EV research as a priority within the domain of Regenerative Medicine, a pivotal area outlined in the Plan. In alignment with this prioritization, NEI organized a workshop inviting twenty experts from within and beyond the visual system. The workshop aimed to review current knowledge in EV research and explore gaps, needs and opportunities for EV research in the eye, including EV biology and applications of EVs in diagnosis, therapy and prognosis within the visual system. This perspective encapsulates the workshop's deliberations, highlighting the current landscape and potential implications of EV research in advancing eye health and addressing visual diseases.

  PublisherOA PDFDOI

• Comparison of EV characterization by commercial high‐sensitivity flow cytometers and a custom single‐molecule flow cytometer

  Journal of Extracellular Vesicles · 2024-08-01 · 39 citations

  articleOpen accessSenior authorCorresponding

  Abstract High‐sensitivity flow cytometers have been developed for multi‐parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high‐sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single‐molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di‐8‐ANEPPS and with PE‐conjugated anti‐EGFR or anti‐tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross‐calibrated, hard‐dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di‐8‐ANEPPS + /PE + particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin‐labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super‐resolution/single‐molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter‐platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter‐instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi‐quantitative LOD values for other flow cytometers.

  PublisherOA PDFDOI

Recent grants

• High-precision mapping of the spatial organization of synaptic-vesicle membrane proteins

  NIH · $1.9M · 2017–2023

• Digital LAMP as a point of care assay for HPV detection

  NIH · $1.9M · 2016–2020

• NIH Grant R21GM103459

  NIH · $378k · 2015

• NIH Grant R21AG029574

  NIH · $491k · 2010

• Emulsion digital PCR for TKI discontinuation studies

  NIH · $1.5M · 2016–2021

Frequent coauthors

• Mei‐Ling Cheng

  Chang Gung Memorial Hospital

  105 shared

• Jiangbo Yu

  79 shared

• Changfeng Wu

  78 shared

• Hung‐Yao Ho

  Chang Gung Memorial Hospital

  73 shared

• Owe Orwar

  44 shared

• Bryant S. Fujimoto

  University of Washington

  41 shared

• Fangmao Ye

  University of Washington

  41 shared

• Perry G. Schiro

  38 shared

Education

• B.A., Neurobiology

  University of California at Berkeley

  1993

• B.S., Chemistry

  University of California at Berkeley

  1993

• Ph.D., Chemistry

  Stanford University

  1998

Awards & honors

• American Chemical Society National Fresenius Award
• Pittcon Achievement Award
• Fellow of the AAAS (2011)
• Keck Distinguished Young Scholar in Biomedical Research
• Alfred P. Sloan fellow