Optogenetics integrated with a variety of genetically encoded methods have been considered a very powerful technique for identifying neurovascular coupling mechanism. Recently, optogenetics has been applied to non-neuronal cells as well as neurons, to study the functions of various organs and tissues such as cardiovascular and bladder. However, the cerebrovascular network has a complicated environment in which cells and tissues with various features and functions coexist. In order to successfully apply optogenetics to study neurovascular coupling mechanism, it is necessary to develop not only optical stimulation techniques but also appropriate monitoring techniques. In addition, it has been very difficult to develop an appropriate optical system, since it is necessary to operate with the neurovascular network of a living animal maintained in the normal condition. Therefore, we have developed a fiber-based all-optical system that enables successful application of optogenetics to study neurovascular coupling mechanisms in living mouse. The developed optical system can perform rapid fluorescence imaging and optical stimulation at the same time, so it can measure various cell specific reactions and rapid blood flow that change after optical stimulation. In this study, we were able to observe changes in blood vessel diameter and blood flows caused by the cell activity of smooth muscle cells after selective optical stimulation using the developed all-optical system in SM22α-ChR2 transgenic mice. Also, the developed all-optical system can be used to study cerebrovascular disease or hemodynamics using optogenetics in vivo.
We present all-optical imaging system for studying cerebral blood flow regulation. To investigate changes in blood flow in the brain, it is necessary to visualize the vascular microstructure between neurons, astrocytes, and vascular cells and monitor changes in vessel diameter and blood flow velocity. Optogenetics is an excellent technology that can be applied to cerebral blood flow regulation studies that require superior spatial and temporal resolution and individual control of cellular activity. The developed optical system is a new integrated optical imaging system that can apply all of the techniques mentioned above for cerebral blood flow regulation research. The optical system includes a dual-color fluorescence imaging system and a laser stimulation system. The system successfully performed dual-color fluorescence imaging with a 50 μm grid pattern in the two wavelength ranges of 515-545 nm and 608-631 nm. It also performed laser stimulation with a minimum output laser pattern size of 2 x 2 μm2 and a maximum intensity of up to 120 mW/mm2. In addition, the optical system measured the fast flow velocity of fluorescent microbeads up to 1.9 μm/ms. Experimental results show that the system can be a promising tool for cerebral blood flow regulation studies that require optogenetic stimulation and dual-fluorescence imaging while measuring blood flow velocity. In the future, the all-optical imaging system will be applied to fiber bundle-based endoscopic systems developed to study cerebral blood flow regulation using optogenetics in living animal brains.