Mohammed, A. I.; Wiesner, D.; Dachs, A.; Wippermann, E.; Goellner, S.; Willeit, S.; Klemm, U.; Schulz, S.; Perleberg, B.; Galligan, J.; Rodrigues, E.; Rembeck, I.; Duerkop, A.; Ntziachristos, V.; Rudack, T.; Stiel, A. C.

Bacteriophytochromes (BphPs) find increasing interest as near-infrared (NIR) labels for imaging. Applications range from whole animal imaging to cell- to super-resolution microscopy. Here we present a comprehensive study of a BphP from Rhizobium etli (ReBphP) allowing mutant-based insights into BphP photophysics. This is complemented by QM/MM-optimized deep-learning structure predictions of ReBphP and variants in their photoswitched states, rationalizing the effects of variants. Pertaining to imaging, based on our study we identify a bright and far red-shifted BphP for imaging in mammalian cells as well as a fluorescent photoswitching BphP. Utilizing the latter, we introduce photoswitching fluorescence background suppression for in vivo whole animal fluorescence imaging achieving higher contrast over background than possible utilizing non-switching labels.