title: In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy.
abstract: Genetically expressed fluorescent proteins have been shown to provide photoacoustic contrast. However, they can be limited by low photoacoustic generation efficiency and low optical absorption at red and near infrared wavelengths, thus limiting their usefulness in mammalian small animal models. In addition, many fluorescent proteins exhibit low photostability due to photobleaching and transient absorption effects. In this study, we explore these issues by synthesizing and characterizing a range of commonly used fluorescent proteins (dsRed, mCherry, mNeptune, mRaspberry, AQ143, E2 Crimson) and novel non-fluorescent chromoproteins (aeCP597 and cjBlue and a non-fluorescent mutant of E2 Crimson). The photoacoustic spectra, photoacoustic generation efficiency and photostability of each fluorescent protein and chromoprotein were measured. Compared to the fluorescent proteins, the chromoproteins were found to exhibit higher photoacoustic generation efficiency due to the absence of radiative relaxation and ground state depopulation, and significantly higher photostability. The feasibility of converting an existing fluorescent protein into a non-fluorescent chromoprotein via mutagenesis was also demonstrated. The chromoprotein mutant exhibited greater photoacoustic signal generation efficiency and better agreement between the photoacoustic and the specific extinction coefficient spectra than the original fluorescent protein. Lastly, the genetic expression of a chromoprotein in mammalian cells was demonstrated. This study suggests that chromoproteins may have potential for providing genetically encoded photoacoustic contrast.
authors.affiliation: Department of Medical Physics and Bioengineering, University College London, Gower Street, London WC1E 6BT, UK ; Julius Wolff Institut, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany ; Contributed equally to this work.
authors.forename: Jan
authors.initials: J
authors.lastname: Laufer
authors.forename: Amit
authors.initials: A
authors.lastname: Jathoul
authors.forename: Martin
authors.initials: M
authors.lastname: Pule
authors.forename: Paul
authors.initials: P
authors.lastname: Beard
keywords: (000.1430) Biology and medicine; (110.5125) Photoacoustics; (300.0300) Spectroscopy