TWO PHOTON ACTIVATION AND IMAGING OF CHR2 EXPRESSING EXCITABLE CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 首次采用近红外激光微束双光子激发研究了通道视紫红质2（ChR 2）在可兴奋细胞中的激活。通过测量钙指示剂染料钙橙子在不同波长处的荧光强度作为双光子激发微束的功率的函数，我们确定了近红外微束的激活电位作为波长的函数。发现双光子激活光谱与单光子激活进行的测量相匹配。然而，双光子激活被发现以非线性的方式增加的双光子激光微束的功率密度。这种方法使我们能够以高空间分辨率激活ChR 2致敏的可兴奋细胞的不同区域。此外，通过使用双光子近红外微束证明了球体细胞模型以及小鼠脑切片中ChR 2的深度激活，这是使用单光子激活不可能的。这种在具有高空间和时间分辨率的遗传靶向细胞中识别、激活和检测ChR 2诱导的细胞激活的全光学方法将提供一种对组织或生物体的特定靶区域进行微创深度激活的新方法，所述组织或生物体的特定靶区域已经通过ChR 2或类似的光可激发分子的遗传靶向而变得光敏。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Two-photon excitation with a near-IR laser micro-beam is used to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time. By measuring the fluorescence intensity of the calcium indicator dye calcium orange at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the near IR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a non-linear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as mouse brain slices was demonstrated by use of the two-photon near-IR microbeam which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.