Fluorescent protein-based biosensors are indispensable molecular tools for life science research. The invention and development of high-fidelity biosensors for a particular molecule or molecular event often catalyze important scientific breakthroughs. Understanding the structural and functional organization of brain activities remain a subject for which optical sensors are in desperate need and of growing interest. Here, we review genetically encoded fluorescent sensors for imaging neuronal

more »

... vities with a focus on the design principles and optimizations of various sensors. New bioluminescent sensors useful for deep-tissue imaging are also discussed. By highlighting the protein engineering efforts and experimental applications of these sensors, we can consequently analyze factors influencing their performance. Finally, we remark on how future developments can fill technological gaps and lead to new discoveries. Chemosensors 2017, 5, 32 2 of 28 clinically important tools for neurosurgical planning and diagnosing [12] . However, these techniques can only provide limited spatiotemporal information about brain function. Complementary to these techniques are electrophysiology and optical neuroimaging, through which electric and neurochemical signals in the brain can be detected and further related to neuronal and cortical functions [13] . While electrophysiology, the 'gold standard' for investigating neuronal functions [14, 15] , can directly measure electrical activity of cells with high sensitivity, the invasive requirement of physical contact with tissues and the accompanied poor spatial resolution lend its dominant position in neuroscience to be challenged by the emerging optical neuroimaging techniques [16] . The use of light to study neuronal signaling has several advantages, including limited invasiveness, tunable wavelength, high spatial resolution, and sensitive detection [17] . Optical neuroimaging measures neuronal activities by converting neuronal signals, such as voltage, calcium ions (Ca 2+ ), and neurotransmitters into light signal outputs (Figure 1) . It is thus an indirect approach towards studying neuronal signaling that necessitates the use of reporters (or probes, sensors, indicators, etc.) [16] . As such, the invention of optical reporters for brain imaging has been a highly intensive area of research [18] . Historically, calcium-[19] and voltage-sensitive dyes [20] have played pivotal roles in optically recording brain signals in vitro and in vivo. The use of dyes, however, is invasive and may result in non-specific staining. Moreover, the spatial and temporal localization of dyes cannot be easily controlled, making targeted or chronic imaging difficult. As a complementary and competing technology, genetically encoded fluorescent indicators are becoming increasingly popular because they (1) can be introduced into cells through transgenic DNA expression; (2) enable targeted expression with precise spatial and temporal control; (3) facilitate large-scale recording of neuronal activities in vitro and in vivo; (4) are amenable to protein engineering and optimizations; (5) are relatively noninvasive; and (6) are suitable for chronic imaging. However, since genetically encoded fluorescent indicators often require genetic delivery, they are less appropriate for direct, in vivo applications on human subjects. The past two decades has witnessed the fast development of a growing list of genetically encoded fluorescence probes, which have found broad applications within the neuroscience community for analyzing neural circuit functions [21] [22] [23] . With an increasing number of probes being successfully applied for circuit interrogation in vivo, it is now evident that they are transforming neuroscience in an unprecedented manner [21] . While reviews for Ca 2+ and voltage sensors are available, very few of them covers genetically encoded indicators for various phenotypic effects of neuronal transmission and evaluate them within a unified framework [24] . To this end, we summarize the history and recent development in the field of optical sensors for neuronal activities, with a focus on indicators for Ca 2+ , voltage, neural transmitters, and pH changes.