Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV

Rachael J. Homans, Raja Khan, Michael B Andrews, Annemette E Kjeldsen, Louise Natrajan, Steven Marsden, Edward Mckenzie, John M Christie, Alex Jones

Research output: Contribution to journalArticlepeer-review


LOV-domains are ubiquitous photosensory proteins that are commonly re-engineered to serve as powerful and versatile fluorescent proteins and optogenetic tools. The photoactive, flavin chromophore, however, is excited using short wavelengths of light in the blue and UV, which have limited penetration into biological samples and can cause photodamage. Here, we have used non-linear spectroscopy and microscopy of the fluorescent protein, iLOV, to reveal that functional variants of LOV can be activated to great effect by two non-resonant photons of lower energy, near infrared light, not only in solution but also in biological samples. The two photon cross section of iLOV has a significantly blue-shifted S0 → S1 transition compared with the one photon absorption spectrum, suggesting preferential population of excited vibronic states. It is highly likely, therefore, that the two photon absorption wavelength of engineered, LOV-based tools is tuneable. We also demonstrate for the first time two photon imaging using iLOV in human epithelial kidney cells. Consequently, two photon absorption by engineered, flavin-based bio-molecular tools can enable non-invasive activation, with high depth resolution and the potential for not only improved image clarity but also enhanced spatiotemporal control for optogenetic applications.
Original languageEnglish
JournalPhysical Chemistry Chemical Physics
Early online date4 Jun 2018
Publication statusPublished - 2018

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology
  • Manchester Cancer Research Centre


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