Research
i. Super-resolution mechanobiology (Tension-PAINT)
Even though mechanical forces play a crucial role in biological processes, imaging cellular force with sub-100nm resolution remains a significant challenge. In response to this, we introduced a technique named Tension Points Accumulation for Imaging in Nanoscale Topography (tPAINT). This method combines molecular tension probes with the DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) technique, allowing us to depict piconewton mechanical events with a resolution of approximately 25nm. To enable live-cell dynamic tension imaging, we designed reversible probes that reveal a hidden docking site only when subjected to forces surpassing a set mechanical threshold (roughly 7–21 pN). Furthermore, we developed a secondary type of tPAINT probe, which permanently uncovers its hidden docking site, thereby recording the force history over time. Although this provides enhanced spatial resolution, it compromises temporal dynamics. We employed both forms of tPAINT probes to visualize integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Notably, tPAINT enabled us to establish a connection between platelet forces at the leading edge of cells and the dynamic actin-rich ring initiated by the Arp2/3 complex.
ii. High-throughput biomolecular force manipulation (Force-jump)
Single-Molecule Force Spectroscopy (SMFS) is a technique wherein a tethered molecule is stretched using a specialized instrument like an Atomic Force Microscope (AFM) or optical tweezer. This is done to observe how macromolecules behave under applied force. However, a challenge with SMFS is its sequential and slow nature as measurements are done one molecule at a time. To overcome this significant challenge, we developed the Polymer Force Clamp (PFC) and the DNA Origami Polymer Force Clamp (OPFC). These tools allow us to manipulate the mechanical forces applied to molecules in parallel, without needing dedicated SMFS instruments or surface tethering. The first-generation PFC incorporates a plasmonic nanorod core enveloped by a thermoresponsive polymer shell. Upon optical heating, the nanorod induces a rapid contraction of the polymer, converting light into mechanical work that unfolds the target molecules. The applied forces' magnitude and duration can be controlled by adjusting the illumination, influencing the degree and duration of particle contraction. Using single-molecule fluorescence imaging, we demonstrated the reproducible mechanical unfolding of DNA hairpins. Further, we were able to trigger 50 different particles in less than a minute, surpassing conventional AFM's speed. Building on this, we developed the second-generation tool, the OPFC, which positions target molecules between a responsive polymer particle that contracts on demand and a rigid nanoscale DNA origami beam. As a demonstration, we documented the steady-state and time-resolved (Force-jump) mechanical unfolding dynamics of DNA hairpins using fluorescence signals from molecule ensembles. Our results were also corroborated through modeling.
iii. DNA-encoding multiplex imaging for spatial biology and cellular sociology
RNA and protein molecules serve as the critical foundation for most biological activities, with their functionality tightly interwoven with their spatial localization and abundance. Fluorescence imaging techniques such as Immunofluorescence imaging and single-molecule fluorescent in situ hybridization (smFISH) have been invaluable in quantifying and spatially mapping protein and RNA molecules, providing insightful answers to many biological queries. However, due to the finite number of fluorophores and secondary antibodies, traditional methods can only profile 1-3 targets simultaneously. To address this demand for multiplexing, DNA-exchange-based and signal amplification methods have been employed. These strategies involve the use of orthogonal DNA probe binding and rapid probe exchange to reveal target molecules, enabling swift, multiplexed target switching for imaging. Yet, several technical challenges persist, limiting their broad adoption. Presently, I'm working alongside numerous trailblazers in the realms of DNA nanoscience and spatial biology, striving to develop the next generation of DNA-based multiplex imaging platforms. These platforms aim to scale proteomic and transcriptomic studies, promising substantial strides in our understanding of complex biological systems.