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We report the potentials of nanometer-sized contrast agents which are called gas vesicles (GVs) for super-resolution ultrasound (SRUS) imaging to diagnose of vasculature deep inside tissue. Thus, we developed the GVs and ultrasound localization microscopy (ULM) based on singular value decomposition and 2D cross-correlation techniques. Furthermore, the SRUS imaging of the vessel-mimicking phantom with the GVs was performed. These results demonstrate that GVs could have potentials as novel contrast agents at nanoscale for implementing the SRUS imaging, thus indicating that ULM with GVs would be used for better visualization of micro-vasculature in vivo. 

The live visualization with fast kinetics of the interaction between cells has been long term challenges because of the lack of efficient stimulation methods. We propose an approach to achieve single cell resolution stimulation and FRET-base calcium live cell imaging to visualize fast kinetics of calcium transport between physically connect neighboring cells. Chemical stimulation stimulates cells within a dish at the same time and is not suitable for the study of cell-cell interaction. We replaced chemical stimulation with ultrasound-based mechanical stimulation approach to provide precise spatiotemporal resolution. To achieve this, we integrated 3D translation stages and epi-fluorescence microscope and a developed 150 MHz high frequency ultrasound with f number of 1 and aperture size of 1 mm. The 150 MHz transducer can focus within 10 micrometers in diameter and directly stimulate cells by disturbing cell plasma membranes without microbubbles. High frequency stimulation was used to introduce calcium ions into cytoplasm of cells. Results demonstrate calcium transport between cells, visualized by FRET calcium biosensor after only one cell was stimulated by the developed high frequency ultrasonic transducer. 

Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10–15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU.