Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
Nat Commun. 2016 Oct 27;7:13268. doi: 10.1038/ncomms13268.
Optogenetic techniques allow intracellular manipulation of Ca by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.
光遗传学技术允许通过照明光吸收探针分子（如通道视紫红质和黑素视蛋白）来对细胞内的 Ca 进行操作。光遗传学刺激的后果最好通过非侵入性的光学方法来记录。然而，目前大多数用于监测 Ca 水平的光学方法都是基于荧光激发的，这可能会导致对光遗传学探针的不必要刺激和其他不期望的影响，如组织自发荧光。发光是一种避免与荧光相关联的问题的替代光学技术。使用一种新的亮荧光素酶，我们在这里开发了一种基于生物发光共振能量转移（BRET）的比率型基因编码 Ca 传感器。该传感器具有很大的动态范围，并与光遗传学探针最佳结合。通过短暂的光脉冲刺激表达黑素视蛋白的培养细胞和表达通道视紫红质的神经元，用光遗传学刺激来量化和成像 BRET Ca 传感器，从而避免了荧光照射的不良后果。
