Analysis of Red-Fluorescent Proteins Provides Insight into Dark-State Conversion and Photodegradation

Abstract

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

“…4 Similar to other FPs, the crystal structure of mCherry reveals excellent interstrand hydrogen-bonding ( e.g ., amide nitrogen-carbonyl oxygen distance < 3 Å), indicative of a rigid beta-barrel structure. However, the well-ordered hydrogen-bonding is disrupted between β-strands 7 and 10 (Figure 1A).…”

“…Tsien and colleagues identified improved FPs by exposing bacterial colonies to a solar illuminator, 16 but this technique is limited to illumination intensities below those necessary for low-copy or single-molecule imaging. Given that photobleaching mechanisms are illumination-dependent, 4 it would be valuable to develop a screening approach in which the excitation intensity could be tuned over a wide range. Additionally, selection within the mammalian context is preferred given that some FPs identified in E. coli fail to fold productively in eukaryotes.…”

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…50,51 RFPs can also suffer from reversible conversion to a transient dark state. 130,131 Protein engineers continue to work on creating ever-better RFPs, yet, to date, an RFP that matches the best avGFP variants in all performance characteristics remains elusive. Nevertheless, we remain confident that such an RFP, or far RFP, will be engineered in the near future.…”

Advances in fluorescence labeling strategies for dynamic cellular imaging

“…However, photostability is highly context dependent and depends upon the excitation intensity, excitation source (laser or lamp), excitation duty-cycle (laser-scanning or wide-field) and excitation duration (microseconds in confocal, tens to hundreds of milliseconds in wide-field). Additionally, FPs convert to dark states, including the triplet and isomerized states, which can be either photoprotective or photoreactive 8 . These experimental difficulties, as well as differences in analysis, render interlaboratory comparisons of photostability challenging.…”

“…In addition to being compatible with the cellular milieu, nontoxic and minimally perturbing, probes need to provide sufficient contrast between the object of interest and background noise. Yet contrast in fluorescence imaging is often influenced by environmental (pH, quenchers, redox agents and O 2 concentration) and experimental (excitation wavelength, intensity, duty-cycle and presence of unbound probe) conditions, necessitating careful attention to detail when selecting a probe for a given application 8 . Additional properties that influence how a probe might be used include photophysical processes such as intersystem crossing, reactive-oxygen sensitization, dark states, quenching and photochromism 9 .…”