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.

Sulfates as chromophores for multiwavelength photoacoustic imaging phantoms

“…This is necessary to ensure a good signal-to-noise ratio (SNR). 17 2. Low propensity for peak power dependent transient effects such as ground-state bleaching/saturation 13,18 (also referred to as resistance to transient photobleaching).…”

Photoacoustic imaging of fluorophores using pump-probe excitation

“…This provides a detection method that is, in principle, limited by the noise floor of the PA image acquisition system. By contrast, the accuracy of fluorophore distributions recovered from multiwavelength images using spectral unmixing methods can also be adversely affected by uncertainties in the a priori information, such as the specific absorption spectra of fluorophores [10]. A further major advantage of this method lies in its simple experimental implementation, which requires the acquisition of two images with and without time delay between the pump and probe pulses, and the calculation of a difference image.…”

“…Second, ground state depopulation, which is caused by long excited state lifetimes, can lead to a transient reduction in the number of molecules in the ground state during ns excitation pulses. This has been shown to result in deviations from the linear dependence of the PA signal amplitude on absorber concentration [23], and in differences between the optical absorption and corresponding PA spectra [10]. Third, intersystem crossing may also reduce the signal amplitude since triplet state relaxation occurs on ms timescales, which does not fulfill the condition of thermal and stress confinement.…”

“…In fluorophores, however, the assumption that the PA spectrum is proportional to the specific absorption spectrum is not always valid. Fluorescence emission and ground state depopulation can reduce the amount of thermalized energy, and hence PA signal amplitude, generated during the typically short (ns), high peak power excitation pulses [10,11]. Since the relative contributions of these effects are also wavelength-dependent, they can compromise the accuracy of spectral unmixing methods.…”

Simultaneous photoacoustic and optically mediated ultrasound microscopy: an in vivo study

“…Meanwhile, the optical absorption spectra of endogenous chromophores (hemoglobin [2], melanin [3], lipids [4], etc.) and exogenous markers (organic dyes [5], nanoparticles [6], fluorescent proteins [7], etc.) enable the corresponding PA imaging to have a level of contrast that is unachievable by the US methods alone [8].…”