In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy
Photoacoustic imaging has been developed as a hybrid biomedical imaging modality where light is utilized to activate the targeted chromophore or fluorophore and an ultrasound transducer is used to detect the resulting thermoelastic expansion. Chromophores and fluorophores absorb photons according to the molar extinction coefficient (ε), where increased absorption of light is proportional to an increase in ε. Following photon absorption electrons are promoted from the ground state to the excited state where they can relax back down to their ground state via emission of a fluorescence photon or vibrational relaxation, which results in heating and thus thermoelastic expansion of the medium. Photoacoustic imaging is advantageous for biomedical imaging because it can provide higher resolution imaging in tissue than optical detection of photons which are multiply scattered in biological media. By contrast, biological tissues scatter ultrasound waves far less than optical photons. However, since laser excitation is required for biomedical applications of photoacoustic imaging, the same principles that govern the desired wavelength range of optical imaging apply. Specifically near infrared (NIR) fluorophores are desirable due to the minimal photon absorption, scattering and autofluorescence in the NIR window as compared to the visible wavelengths. Interestingly, for photoacoustic imaging the properties of the necessary chromophores or fluorophores are opposite those of NIR fluorescence detection. Thus, fluorophores with high quantum yield, or efficient conversion of absorbed photons to fluorescent photons, are undesirable as this competes with the heat deposition and thermoelastic expansion of tissue necessary for photoacoustic detection.
The work by Laufer et al is focused on the in vitro characterization of genetically expressed chromophores for photoacoustic imaging and their mutants to create red shifted chromophores that can be genetically encoded. The use of fluorescent proteins for photoacoustic imaging is challenging because a protein that both acts as a chromophore, with high ε and low quantum yield, and has NIR excitation does not currently exist. The authors characterized a range of commonly used fluorescent proteins including dsRed, mCherry, mNeptune, mRaspberry, AQ143, and E2 Crimson as well as three non-fluorescent novel chromoproteins for their photoacoustic spectra, photoacoustic generation efficiency and photostability. The chromoproteins were found to exhibit improved photoacoustic signal generation in comparison to the fluorescent proteins attributed to the absence of radiative relaxation and ground state depopulation in the chromoproteins as well as significantly higher photostability in the chromoproteins as compared to the fluorescent proteins. The authors demonstrated the feasibility of converting an existing fluorescent protein to a non-fluorescent chromoprotein through their work with E2 Crimson mutagenesis. As proof of concept the authors genetically express the chromoproteins in mammalian cells and measured their photoacoustic spectra, demonstrating that chromoproteins are a feasible photoacoustic contrast option for photoacoustic spectroscopy and imaging in mammalian cells.