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© 2020 The Author(s) For large brain volume manipulations using optogenetics, both effective opsin excitation and efficient light delivery with minimal light absorption are required to minimize the illuminating light intensity and concomitant off-target effects. ArchT, a widely used potent inhibitory opsin, is commonly activated by 532-nm light, which lies on its in vitro excitation peak. However, 532-nm light also lies on a peak range of the hemoglobin absorption spectrum. Therefore, we predicted that 594-nm light is superior in suppressing distant ArchT-expressing neurons, which is slightly off the ArchT-excitation-plateau and largely off the peak of the hemoglobin absorption spectrum. We quantitatively tested this prediction by the electrophysiological recording of the rat cortex in vivo. At illumination distances greater than 500 μm, 594-nm light was more effective than 532-nm light. Its superiority increased with distance. These results validate our prediction and highlight the significance of excitation-absorption trade-off in selecting illumination wavelength for optogenetics in vivo.

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