1. Can you please briefly summarize the paper?

This paper presents a two-color single-molecule fluorescence strategy to quantify actin cargo in individual extracellular vesicles (EVs) secreted by human iPSC-derived cells. We first employ hiPSCs with endogenous Actin-EGFP via CRISPR-Cas9 knock-in (Figure a), and then label the EV membrane with micro red fluorescent protein marker for two-color detection (Figure b). Then we combine fluorescence cross-correlation spectroscopy (FCCS) with coincident fluorescence-burst analysis to reliably separate true cargo-loaded EVs from free fluorescent background (Figure c). With this approach, we measure the fraction of EVs carrying actin, estimate the number of actin molecules per EV at the single-vesicle level, and use the coincident-burst rate as a proxy for changes in loaded-EV secretion dynamics. Biologically, we find that stress-like conditions such as partial differentiated or amyloid-β exposure, are associated with an increased occurrence of actin-loaded EV events compared with undifferentiated case, suggesting that cell state modulates cytoskeletal cargo packaging into EVs. Our platform provides a quantitative, highly specific way to track cytoskeletal disruption through EV cargo and to reveal molecular shifts during neuronal differentiation so that it can provide insights that are essential for advancing therapeutic strategies for neurological disorders.

Figure (a) Generation of human induced pluripotent stem cells (hiPSCs) stably expressing actin-EGFP.  (b) Workflow of EV production, isolation, and staining. hiPSC-actin-EGFP cells are cultured under undifferentiated, partially differentiated, or amyloid-β-treated conditions. (c) Optical setup for dual-color fluorescence detection using time-resolved FCCS.

2. Can you please tell us the main difficulties you had in the laboratory work and how you overcame them?

In this work, the main difficulties were making sure the signals we measured truly came from single, cargo-loaded EVs rather than from free fluorescent proteins, dye aggregates, or random overlaps, and then keeping the results consistent across days. At first, background fluorescence and occasional aggregation made the data look noisy or obscured, so we tightened the workflow step by step: we relied on a dual-label strategy (membrane label plus endogenous actin–EGFP cargo) and only accepted time-coincident events as true loaded EVs. On the instrument side, we treated alignment and cross-talk as a constant risk, so we routinely calibrated with reference samples and kept conservative detection thresholds. With these changes, the measurements became much more robust and cleaner separation of real EV events from background, improved reproducibility, and enough confident statistics to compare secretion/loading differences across cell conditions.

3. Please introduce your laboratory, university or organization to bio-researchers in Korea.

I am working in BioNanoPhotonics Laboratory at Sungkyunkwan University (SKKU), led by Prof. Inki Kim, is part of the Department of Biophysics and the Institute of Quantum Biophysics (IQB). Our group conducts theoretical, computational, and experimental research at the intersection of nanophotonics and biophotonics, aiming to build artificial nanophotonic platforms that can address important challenges in both fundamental science and applied engineering. In particular, we work on areas such as plasmonics, dielectrics, metamaterials, nanofabrication, biophotonics, quantum biophysics, and integrated quantum medical devices, with the broader goal of developing compact, high-performance optical devices and highly sensitive bio-diagnostic technologies.

4. Please tell us your experiences and your thoughts related to research activities abroad.

I’ve had the chance to work in an international research environment, where teams are diverse in background and expertise. What I value most is the very open, discussion-driven culture-people challenged ideas directly but constructively, and that make the science sharper. It also trained me to communicate clearly across disciplines, document my work carefully, and collaborate efficiently. Overall, doing research abroad broadened my perspective and made me more adaptable and confident in leading or contributing to global projects.

5. Can you provide some advice for younger scientists who have plans to study abroad?

A clear purpose is essential when you plan to study abroad. Firstly, define skills you aim to gain and the research direction you want to develop. Importantly, don’t underestimate yourself too much, and don’t wait until everything feels “perfect” to apply. It is helpful to research potential groups carefully and confirm that the laboratory’s expertise, facilities, and research topics align with your goals. Then contact potential supervisors early with a concise, well-prepared introduction, and continue strengthening communication skills in scientific writing, presentations, and discussion. After joining the program, stay proactive by asking questions, keeping thorough documentation, and building professional connections within the research community. Beyond academic outcomes, studying abroad can also develop independence, adaptability, and strong international collaboration skills.

6. Future plan?

My future plan is to keep developing quantitative single-particle/molecule bioanalysis methods and translate them into practical diagnostic tools. I would like to combine advanced optical/nanophotonic technology with real biological questions, build a more robust and user-friendly platform, and eventually help create compact devices that can support early detection and treatment monitoring in healthcare.

7. Do you have anything else that you would like to tell Korean scientists and students?

I just want to say thank you for the strong research culture in Korea. People work seriously, share knowledge, and aim for high standards, and that really pushes science forward. To Korean scientists and students, I hope you keep that strength, but also protect your curiosity and your balance: ask bold questions, collaborate across fields, and don’t be afraid to connect with researchers from other countries. I’d be very happy to contribute and to build bridges between Korean research and the global community, including Vietnam.