RESEARCH

The intricate network of immune cells and diverse biomolecules that summon, rile, calm, and transform these cells create a complex puzzle. Solving this puzzle requires sensitive tools at the molecular and nanoscale level. The Ying Hu Group specializes in developing single-molecule and superresolution bioanalytical techniques to study communications within and between immune cells. The multidisciplinary research combines single-molecule and superresolution microscopy, nanotechnology, biophysical chemistry, and immunobiology.

1. Technology development: Pioneering fluorescent labeling technologies to enhance superresolution imaging, focusing on quantitative and live-cell applications.

The problem: Superresolution imaging is transforming biomedical research by surpassing the diffraction limit of visible light, first defined by Ernst Abbe in 1873. However, as resolution improves, significant technical challenges arise. These include artifacts from immunofluorescent labeling, inaccuracies in molecular quantification, and limitations in quantitative live imaging, all of which have constrained the full potential of superresolution techniques.

Our approach: We have developed a technique collectively termed single-molecule labeling (ACS Nano 2022, Bioconjug Chem 2023, Biophys J 2024). By imaging and deconvolving individual fluorescent labels as they bind to their targets, we developed techniques that improve molecular quantification and minimize artifacts commonly associated with traditional immunofluorescence labeling. Our current work focuses on moving towards small molecule probes for cell labeling and imaging.

2. Nanoscale Immunobiophysics: Leveraging superresolution imaging to decode how immune cells transmit information at and from the plasma membrane in healthy and diseased states, aiming to uncover potential therapeutic targets and mechanisms.

The problem: The dynamic nature of the immune cell membrane plays a crucial role in regulating the exchange of information within and between cells. For example, the immune cells can engulf and neutralize foreign invaders using their plasma membrane and communicate with each other through specialized membrane structures. However, the dynamic and fine features of the plasma membrane are difficult to capture and quantify. Yet, such information is vital for unraveling membrane regulation mechanisms and transient membrane events within physiological or pathological contexts.

Our approach: We utilized single-particle tracking of engineered nanoparticles to map out the topology of a portion of the T cell plasma membrane in 3D (Nanoscale 2021). Subsequently, we revealed the extended membrane fiber networks and migrasomes dendritic cells utilized for antigen uptake, storage, and release (Nano Lett 2022). We further revealed the involvement of F-actin in these membrane fibers (Biophys J 2024, Gunasekara). Our current work focuses on: 1) understanding how cytokines, used by immune cells to communicate with other cells, are stored, transported, and released from T cells (Biophys J 2024, Saed), and 2) understanding membrane signaling events in specialized membrane protrusions.

• H Gunasekara , T Perera, CJ Chao, J Bruno , B Saed, J Anderson, Z Zhao, YS Hu. Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin. Biophys J. 2024 PMID: 38961624. Link.
• B Saed, NT Ramseier, T Perera, J Anderson, J Burnett, H Gunasekara, A Burgess, H Jing, YS Hu, Increased Vesicular Dynamics and Nanoscale Clustering of IL-2 after T Cell Activation. Biophys J. 2024. PMID: 38532626. Link.
• H Jing, B Saed, M Pálmai, H Gunasekara, PT Snee, YS Hu, Fluorescent artificial antigens revealed extended membrane networks utilized by live dendritic cells for antigen uptake, Nano Lett, 2022. PMID: 35499493 pdf Link
• H Jing, M Pálmai, B Saed, A George, PT Snee, YS Hu, Cytosolic delivery of membrane-penetrating QDs into T cell lymphocytes: implications in immunotherapy and drug delivery, Nanoscale, 2021. PMID: 33688882 pdf Link