Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their "darkness," and their ability to act as a FRET acceptor for mEGFP in Hela cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP-REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by ∼50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFPmRFP or mEGFP-original REACh pair by ∼50 %. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP-sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein-protein interactions in individual dendritic spines in brain slices with high sensitivity.

This article is protected by copyright of Korean Journal of Anesthesiology. All rights reserved. Emergence agitation (EA), which is also referred to as emergence delirium, can lead to clinically significant consequences. The mechanism of EA remains unclear. Proposed contributors to EA include age, male sex, type of surgery, emergency operation, use of inhalational anesthetics with low blood-gas partition coefficients, long duration of surgery, anticholinergics, premedication with benzodiazepines, voiding urgency, postoperative pain, and the presence of invasive devices. If preor intraoperative objective monitoring could predict the occurrence of agitation during emergence, this would help to reduce the adverse consequences of EA. Several tools are available for assessing EA; however, its incidence varies considerably according to the assessment tool and definition of EA used, due to the absence of standardized clinical research practice guidelines. Total intravenous anesthesia, propofol, μ-opioid agonists, N-methyl-D-aspartate receptor antagonists, nefopam, α2adrenoreceptor agonists, regional analgesia, multimodal analgesia, parent-present induction, and preoperative education for surgery may contribute to prevention of EA. However, it is difficult to identify patients at high risk for EA and to properly apply EA prevention methods in various clinical situations, because both risk factors and preventive strategies often show inconsistent results depending on the methodology of the study and the patients assessed. This review discusses the most important research topics related to EA and directions for future research.