Dual excitation-emission fluorescence/contractility system

所要求的设备将支持三个研究项目：1)巨噬细胞融合的机制，导致形成多核细胞，如骨吸收的破骨细胞和免疫反应中的巨细胞；2)调节血糖过高时分泌胰岛素的胰腺ß-细胞的葡萄糖转运蛋白活性；3)骨骼肌运动和疲劳时ATP敏感钾通道的保护机制。所有这三个研究首先需要一个装置来量化活细胞中细胞内离子的荧光测量。更具体地说，我们需要i)用于巨噬细胞研究的成像系统和ii)光电倍增管（pmt）来测量ß-细胞和单个肌肉纤维中的总荧光。此外，当用荧光指示剂fura-2测量细胞内钙时，由于收缩持续时间少于200毫秒，因此该装置必须具有在毫秒内在两个激发波长之间切换的能力。该系统还必须能够用SNARF测量细胞内氢离子的两个不同波长的发射光。最后，对于涉及肌肉的研究，我们的单一纤维是在消化胶原蛋白后分离出来的；因此，这些纤维不能连接到力传感器上，因为它们没有肌腱。因此，我们测量肌节缩短作为收缩性的指标。IonOptix双激发-发射荧光/收缩性系统包括一个在两个发射波长之间交替的超开关，两个测量总荧光的pmt，两个相机，一个用于成像分析，另一个用于肌节缩短。该系统是高度通用的，允许通过附带的软件在成像系统和pmt之间切换，或同时测量细胞内离子和肌节长度。

The requested equipment will support three research programs: 1) the mechanism of macrophage fusion that leads to the formation of multinucleated cells such as osteoclasts for bone resorption and giant cells during an immune response; 2) the regulation of the glucose transporter activity in pancreatic ß-cells, which secrete insulin when plasma glucose is too high; and, 3) the protective mechanism of the ATP sensitive potassium channel during exercise and fatigue in skeletal muscle.     All three studies first require a setup that quantifies fluorescence for the measurements of intracellular ion in living cells. More specifically, we need i) an imaging system for the studies involving macrophages and ii) photomultiplier tubes (PMTs) to measure total fluorescence in ß-cells and single muscle fibers. Furthermore, the setup must have the capability to switch between two excitation wavelengths within msec when intracellular calcium is measured with fura-2, a fluorescent indicator, because contractions last less than 200 msec. The system must also be capable of measuring emission light at two different wavelengths for intracellular hydrogen ion with SNARF. Finally, for the study involving muscles, our single fibers are isolated after a digestion of collagen; so, these fibers cannot be attached to a force transducer because they have no tendon left. We therefore measure sarcomere shortening as an index of contractility.     The IonOptix dual excitation-emission fluorescence / contractility system comprise a hyperswitch that alternate between two emission wavelengths, two PMTs to measure total fluorescence, two cameras one for the imaging analysis and another for sarcomere shortening. The system is highly versatile allowing through the included software to switch between the imaging system and PMTs or to simultaneously measure intracellular ion and sarcomere length.