Department of Neurobiology, Stanford University Medical Center, Stanford, CA, USA.
Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Nat Methods. 2023 Jul;20(7):1104-1113. doi: 10.1038/s41592-023-01913-z. Epub 2023 Jul 6.
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
基因编码电压指示剂（GEVI）能够对大脑中的电信号进行光学记录，提供了钙指示剂无法实现的亚阈值灵敏度和时间分辨率。然而，目前还没有报道过使用相同的 GEVIs 进行长时间的单光子和双光子电压成像。在这里，我们报告了对 ASAP 家族 GEVIs 的工程改造，通过反转荧光-电压关系来增强其光稳定性。其中两种 GEVIs，ASAP4b 和 ASAP4e，在 100mV 的去极化时，荧光增加≥180%，而亲本 ASAP3 的荧光减少 50%。使用标准显微镜设备，ASAP4e 能够在数分钟内检测小鼠中的单个尖峰。与之前用于单光子电压记录的 GEVIs 不同，ASAP4b 和 ASAP4e 在双光子激发下也能很好地工作。通过同时成像电压和钙，我们表明 ASAP4b 和 ASAP4e 可以识别位置细胞，并以比常用钙指示剂更高的时间分辨率检测电压尖峰。因此，ASAP4b 和 ASAP4e 扩展了电压成像的功能，使其能够应用于标准的单光子和双光子显微镜，同时延长了电压记录的时间。
