Development of 5′‐ and 7′‐Substituted Luciferin Analogues as Acid‐Tolerant Substrates of Firefly Luciferase

Leaving the light on: We have developed acid-tolerant luciferin analogues with pH-independent luminescence spectra as well as stable luminescence intensity in response to acidic pH change in the presence of luciferase. These compounds permitted robust bioluminescence microscopic imaging of luciferase-expressing single cells in an acidic environment typical of those encountered in many biological processes.