| Autor: |
Yoon S; Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, IN 46556, USA., Pan Y; Department of Bioengineering, University of California, San Diego, CA 92092, USA., Shung K; Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA., Wang Y; Department of Bioengineering, University of California, San Diego, CA 92092, USA. |
| Abstrakt: |
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 Ca 2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca 2+ biosensor with ECFP and YPet (YC3.6 FRET Ca 2+ 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. |
