Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV

Homans, R. J., Khan, R. U., Andrews, M. B., Kjeldsen, A. E., Natrajan, L. S., Marsden, S., McKenzie, E. A., Christie, J. M. and Jones, A. R. (2018) Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV. Physical Chemistry Chemical Physics, 20(25), pp. 16949-16955. (doi: 10.1039/c8cp01699b) (PMID:29873653)

[img]
Preview
Text
163735.pdf - Accepted Version

1MB

Abstract

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 regions, 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Christie, Professor John and Kjeldsen, Miss Annemette
Authors: Homans, R. J., Khan, R. U., Andrews, M. B., Kjeldsen, A. E., Natrajan, L. S., Marsden, S., McKenzie, E. A., Christie, J. M., and Jones, A. R.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Physical Chemistry Chemical Physics
Publisher:Royal Society of Chemistry
ISSN:1463-9076
ISSN (Online):1463-9084
Published Online:04 June 2018
Copyright Holders:Copyright © 2018 the Owner Societies
First Published:First published in Physical Chemistry Chemical Physics 20(25): 16949-16955
Publisher Policy:Reproduced in accordance with the publisher copyright policy

University Staff: Request a correction | Enlighten Editors: Update this record

Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
659801Photoreceptor Engineering to Modulate Plant GrowthJohn ChristieBiotechnology and Biological Sciences Research Council (BBSRC)BB/M002128/1RI MOLECULAR CELL & SYSTEMS BIOLOGY