Although fluorescent indicators have been broadly utilized for monitoring bioactivities, fluorescence imaging, when applied to mammals, is limited to superficial targets or requires invasive surgical procedures. Thus, there is emerging interest in developing bioluminescent indicators for noninvasive mammalian imaging. Bioluminescence imaging (BLI) of neuronal activity is highly desired but hindered by insufficient photons needed to digitalize fast brain activities. In this work, we develop a luciferase prosubstrate deliverable at an increased dose and activated in vivo by nonspecific esterase. We further engineer a bright, bioluminescent indicator with robust responsiveness to calcium ions (Ca²⁺) and appreciable emission above 600 nm. Integration of these advantageous components enables the imaging of the activity of neuronal ensembles in awake mice minimally invasively with excellent signal-to-background and subsecond temporal resolution. This study thus establishes a paradigm for studying brain function in health and disease.

尽管荧光指示剂已广泛用于监测生物活性，但荧光成像在应用于哺乳动物时仅限于浅表目标或需要侵入性外科手术。因此，人们对开发用于非侵入性哺乳动物成像的生物发光指示器产生了兴趣。神经元活动的生物发光成像（BLI）是非常需要的，但由于数字化快速大脑活动所需的光子不足而受到阻碍。在这项工作中，我们开发了一种荧光素酶原底物，可增加剂量并在体内被非特异性酯酶激活。我们进一步设计了一种明亮的生物发光指示剂，对钙离子 (Ca ²⁺ ) 具有强大的响应能力，并且在 600 nm 以上具有明显的发射。这些有利组件的集成使得能够以微创方式对清醒小鼠的神经元群的活动进行成像，并具有出色的信号背景和亚秒时间分辨率。因此，这项研究为研究健康和疾病中的大脑功能建立了一个范例。