An optimal genetically-encoded probe for photoacoustic (PA) imaging should exhibit high optical absorption, low fluorescence quantum yield, and an absorption maxima within the near-infrared (NIR) window. One promising candidate is a newly engineered chromoprotein (CP), designated dark small ultra-red fluorescent protein (dsmURFP), which is based on a cyanobacterial phycobiliprotein. To optimize dsmURFP characteristics for PA imaging, we have developed a directed evolution method to iteratively screen libraries of protein variants with three different screening systems. Firstly, we took inspiration from dark-acceptor (also known as dark-quencher)-based Förster resonance energy transfer (FRET) constructs, and used dsmURFP as a dark acceptor from a mCardinal fluorescent donor. The rationale for this design was that the higher the extinction coefficient of the dsmURFP, the more the emission of the donor would be quenched. In addition, more energy transferred to the dark acceptor would lead to more thermoelastic expansion and a stronger PA signal. Three rounds of evolution using this first strategy resulted in dsmURFP1.3 that quenched the emission of mCardinal ~2-fold more efficiently than dsmURFP. Secondly, an absorption-based screening based on visual inspection of plates led to identification of the variant dsmURFP1.4, which exhibited a 2-fold higher absorbance and a 5 nm red shift. Thirdly, we developed a colony-based photoacoustic screening method. To demonstrate the utility of our optimized variants, we used photoacoustic imaging to visualize dsmURFP and its variants in phantom and in vivo experiments using chicken embryo models and murine bacterial bladder infection models.
Fluorescent proteins are the most common and versatile class of genetically encoded optical probes. While structure-guided
rational design and directed evolution approaches have largely overcome early problems such as oligomerization,
poor folding at physiological temperatures, and availability of wavelengths suitable for multi-color imaging, nearly all
fluorescent proteins have yet to be fully optimized. We have developed novel methods for evaluating the current
generation of fluorescent proteins and improving their remaining suboptimal properties. Little is yet known about the
mechanisms responsible for photobleaching of fluorescent proteins, and inadequate photostability is a chief complaint
among end users. In order to compare the performance of fluorescent proteins across the visual spectrum, we have
standardized a method used to measure photostability in live cells under both widefield and confocal laser illumination.
This method has allowed us to evaluate a large number of commonly used fluorescent proteins, and has uncovered
surprisingly complex and unpredictable behaviors in many of these proteins. We have also developed novel methods for
selecting explicitly for high photostability during the directed evolution process, leading to the development of highly
improved monomeric orange and red fluorescent proteins. These proteins, most notably our photostable derivative of
TagRFP, have remarkably high photostability and have proven useful as fusion tags for long-term imaging. Our methods
should be applicable to any of the large number of fluorescent proteins still in need of improved photostability.
Green fluorescent protein variants have been developed that report real-time change in pH and redox potential in living cells. The variants involve cysteine substitutions near the chromophore, which greatly alter the sensitivity of the protein to changes in its environment. Measurements can be made on single living cells in the fluorescence microscope or in cell suspension with an ordinary fluorimeter. The indicators are ratiometric by emission and/or excitation, which means that measurements at two different wavelengths are sufficient to determine both the quantity being measured and the indicator GFP concentration. The photophysics of a novel blue/green dual emission GFP variant will be presented.
The design principles, crystal structures and ultrafast spectroscopic analysis of probe response will be discussed in terms of atomic models involving excited state proton transfer. Some applications in living cells will be presented.
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