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Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV.
- Source :
-
Physical chemistry chemical physics : PCCP [Phys Chem Chem Phys] 2018 Jun 27; Vol. 20 (25), pp. 16949-16955. - Publication Year :
- 2018
-
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.
- Subjects :
- Escherichia coli
Flavins chemistry
Flavoproteins genetics
HEK293 Cells
Humans
Infrared Rays
Microscopy, Fluorescence, Multiphoton methods
Photons
Protein Conformation
Spectrometry, Fluorescence methods
Flavoproteins chemistry
Fluorescent Dyes chemistry
Luminescent Proteins chemistry
Optical Imaging methods
Subjects
Details
- Language :
- English
- ISSN :
- 1463-9084
- Volume :
- 20
- Issue :
- 25
- Database :
- MEDLINE
- Journal :
- Physical chemistry chemical physics : PCCP
- Publication Type :
- Academic Journal
- Accession number :
- 29873653
- Full Text :
- https://doi.org/10.1039/c8cp01699b