Background

Brain visual circuits are often studied in vivo by imaging Ca²⁺ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators´ emission spectra, resulting in significant contamination of calcium signals.

New method

To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca²⁺ indicator´s emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials –VEPs) and 3D acousto-optic two-photon (2P) population Ca²⁺ imaging in mouse primary visual cortex (V1).

Results

VEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca²⁺ imaging in a Thy1 GP4.3 line revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable.

Comparison with existing methods

Masking or shielding the microscope are often used to minimize the contamination of Ca²⁺ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding.

Conclusions

Together, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca²⁺ imaging with electrophysiology and optogenetics.

中文翻译：

---

蓝光波长光的视觉刺激驱动 V1 有效消除双光子钙成像过程中的杂散光污染

背景

通常通过用绿移发射光谱对 Ca ²⁺指示剂进行成像来在体内研究大脑视觉回路。多色白色视觉刺激的光谱与指标的发射光谱部分重叠，导致钙信号受到严重污染。

新方法

为了克服光污染问题，我们选择蓝色视觉刺激，其光谱成分与 Ca ²⁺指示剂的发射光谱不重叠。为了比较对蓝色和白色刺激的视觉反应，我们在小鼠初级视觉皮层 (V1) 中使用了电生理学（视觉诱发电位 –VEP）和 3D 声光双光子 (2P) 群体 Ca ²⁺成像。

结果

响应蓝色和白色刺激的 VEP 具有可比的峰值幅度和延迟。在 Thy1 GP4.3 线中的Ca ²⁺成像显示，对蓝色和白色刺激作出反应的神经元群体在很大程度上是重叠的，它们的反应具有相似的幅度，并且功能反应特性（例如方向和方向选择性）也具有可比性。

与现有方法的比较

通常使用掩蔽或屏蔽显微镜来最大限度地减少白光对 Ca ²⁺信号的污染，但它们耗时、体积大，因此会限制实验设计，尤其是在越来越频繁使用的清醒设置中。不干扰成像的蓝色刺激允许省略屏蔽。

结论

总之，我们的结果表明，选定的蓝光刺激引起的反应与小鼠 V1 中白色刺激引起的反应相当。这将使行为设置中成像实验的复杂设计更容易，并促进 Ca ²⁺成像与电生理学和光遗传学的结合。