Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, United States of America.
J Neural Eng. 2017 Oct;14(5):056018. doi: 10.1088/1741-2552/aa7ded.
Virus-transduced, intracellular-calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. Due to the existence of an optimal time window for the expression of calcium indicators, a suitable tool for tracking GECI expression in vivo following transduction is highly desirable.
We developed a noninvasive imaging approach based on a custom-modified, low-cost fundus viewing system that allowed us to monitor and characterize in vivo bright-field and fluorescence images of the mouse retina. AAV2-CAG-GCaMP6f was injected into a mouse eye. The fundus imaging system was used to measure fluorescence at several time points post injection. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array and used calcium imaging to evaluate the responsiveness of retinal ganglion cells (RGCs) to external electrical stimulation.
The noninvasive fundus imaging system clearly resolves individual (RGCs and axons. RGC fluorescence intensity and the number of observable fluorescent cells show a similar rising trend from week 1 to week 3 after viral injection, indicating a consistent increase of GCaMP6f expression. Analysis of the in vivo fluorescence intensity trend and in vitro neurophysiological responsiveness shows that the slope of intensity versus days post injection can be used to estimate the optimal time for calcium imaging of RGCs in response to external electrical stimulation.
The proposed fundus imaging system enables high-resolution digital fundus imaging in the mouse eye, based on off-the-shelf components. The long-term tracking experiment with in vitro calcium imaging validation demonstrates the system can serve as a powerful tool monitoring the level of genetically-encoded calcium indicator expression, further determining the optimal time window for following experiment.
病毒转导的细胞内钙指示剂是神经活动的有效报告者，具有细胞特异性标记的优势。由于钙指示剂表达的最佳时间窗口的存在，因此非常需要一种用于跟踪转导后体内 GECI 表达的合适工具。
我们开发了一种非侵入性成像方法，该方法基于经过定制的低成本眼底观察系统，该系统使我们能够监测和表征活体小鼠视网膜的明场和荧光图像。将 AAV2-CAG-GCaMP6f 注射到一只小鼠的眼睛中。使用眼底成像系统在注射后的多个时间点测量荧光。在定义的时间点，我们准备了在透明多电极阵列上安装的全视网膜，并使用钙成像来评估视网膜神经节细胞（RGC）对外部电刺激的反应性。
非侵入性眼底成像系统可清晰分辨单个（RGCs 和轴突。RGC 荧光强度和可观察到的荧光细胞数量从病毒注射后第 1 周到第 3 周呈相似的上升趋势，表明 GCaMP6f 表达持续增加。对体内荧光强度趋势和体外神经生理反应性的分析表明，强度与注射后天数的斜率可用于估计外部电刺激下 RGC 钙成像的最佳时间。
所提出的眼底成像系统基于现成的组件，可实现小鼠眼部的高分辨率数字眼底成像。通过体外钙成像验证的长期跟踪实验表明，该系统可以作为一种强大的工具，用于监测基因编码钙指示剂表达水平，进一步确定后续实验的最佳时间窗口。
