Abstract:Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form d… Show more
“…2). Such FL shortening in cellular environment has been described for EGFP and some other FPs 5,14,15 . Also shifts in experimental lifetime values of the same fluorophore may be attributed to the different hardware sets used.Figure 2Fluorescence and fluorescence lifetime imaging microscopy of live HeLa cells expressing EGFP-actin (mainly in cytoplasm; Tm ~2.2 ns), EGFP-T65G-histone 2B (in nucleus; Tm ~1.1 ns), BrUSLEE-mito (in mitochondria; Tm ~0.8 ns).…”
Section: Resultsmentioning
confidence: 59%
“…A significant progress has been achieved in development of FPs with long fluorescence lifetimes, e.g., cyan mTurquoise2 (4.0 ns) 6 , green WasCFP and NowGFP (5.1 ns) 5,7 , red mScarlet (3.9 ns) 8 . At the same time, a field of FPs with subnanosecond lifetimes remains almost unexplored.…”
Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay rate at every pixel of an image. FLIM can separate probes of the same color but different fluorescence lifetimes (FL), thus it is a promising approach for multiparameter imaging. However, available GFP-like fluorescent proteins (FP) possess a narrow range of FLs (commonly, 2.3–3.5 ns) which limits their applicability for multiparameter FLIM. Here we report a new FP probe showing both subnanosecond fluorescence lifetime and exceptional fluorescence brightness (80% of EGFP). To design this probe we applied semi-rational amino acid substitutions selection. Critical positions (Thr65, Tyr145, Phe165) were altered based on previously reported effect on FL or excited state electron transfer. The resulting EGFP triple mutant, BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), allows for both reliable detection of the probe and recording FL signal clearly distinguishable from that of the spectrally similar commonly used GFPs. We demonstrated high performance of this probe in multiparameter FLIM experiment. We suggest that amino acid substitutions described here lead to a significant shift in radiative and non-radiative excited state processes equilibrium.
“…2). Such FL shortening in cellular environment has been described for EGFP and some other FPs 5,14,15 . Also shifts in experimental lifetime values of the same fluorophore may be attributed to the different hardware sets used.Figure 2Fluorescence and fluorescence lifetime imaging microscopy of live HeLa cells expressing EGFP-actin (mainly in cytoplasm; Tm ~2.2 ns), EGFP-T65G-histone 2B (in nucleus; Tm ~1.1 ns), BrUSLEE-mito (in mitochondria; Tm ~0.8 ns).…”
Section: Resultsmentioning
confidence: 59%
“…A significant progress has been achieved in development of FPs with long fluorescence lifetimes, e.g., cyan mTurquoise2 (4.0 ns) 6 , green WasCFP and NowGFP (5.1 ns) 5,7 , red mScarlet (3.9 ns) 8 . At the same time, a field of FPs with subnanosecond lifetimes remains almost unexplored.…”
Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay rate at every pixel of an image. FLIM can separate probes of the same color but different fluorescence lifetimes (FL), thus it is a promising approach for multiparameter imaging. However, available GFP-like fluorescent proteins (FP) possess a narrow range of FLs (commonly, 2.3–3.5 ns) which limits their applicability for multiparameter FLIM. Here we report a new FP probe showing both subnanosecond fluorescence lifetime and exceptional fluorescence brightness (80% of EGFP). To design this probe we applied semi-rational amino acid substitutions selection. Critical positions (Thr65, Tyr145, Phe165) were altered based on previously reported effect on FL or excited state electron transfer. The resulting EGFP triple mutant, BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), allows for both reliable detection of the probe and recording FL signal clearly distinguishable from that of the spectrally similar commonly used GFPs. We demonstrated high performance of this probe in multiparameter FLIM experiment. We suggest that amino acid substitutions described here lead to a significant shift in radiative and non-radiative excited state processes equilibrium.
“…SEM images were obtained by Hitachi S-4800. Time-resolved photoluminescence (TRPL) curves were recorded using a commercial Time-Correlated Single Photon Counting (TCSPC) system (FluoTim 200, PicoQuant)37. Samples were photoexcited by picosecond diode laser of 670 nm (LDH-P-C-670, PicoQuant) with a variable repetition rate (1 MHz).…”
In CH3NH3PbI3-based high efficiency perovskite solar cells (PSCs), tiny amount of PbI2 impurity was often found with the perovskite crystal. However, for two-step solution process-based perovskite films, most of findings have been based on the films having different morphologies between with and without PbI2. This was mainly due to the inferior morphology of pure perovskite film without PbI2, inevitably produced when the remaining PbI2 forced to be converted to perovskite, so advantages of pure perovskite photoactive layer without PbI2 impurity have been overlooked. In this work, we designed a printing-based two-step process, which could not only generate pure perovskite crystal without PbI2, but also provide uniform and full surface coverage perovskite film, of which nanoscale morphology was comparable to that prepared by conventional two-step solution process having residual PbI2. Our results showed that, in two-step solution process-based PSC, pure perovskite had better photon absorption and longer carrier lifetime, leading to superior photocurrent generation with higher power conversion efficiency. Furthermore, this process was further applicable to prepare mixed phase pure perovskite crystal without PbI2 impurity, and we showed that the additional merits such as extended absorption to longer wavelength, increased carrier lifetime and reduced carrier recombination could be secured.
“…The p K a of some of the most commonly used FPs (e.g. EGFP – p K a EGFP = 6.0, sfGFP – p K a sfGFP = 5.9, EYFP – p K a EYFP = 6.9) suggests that they are less than ideal to track proteins in acidic environments (Miyawaki, Griesbeck, Heim, & Tsien, ; Roberts et al., ; Sarkisyan et al., ). Moreover, p K a measurements are performed in vitro and may not reflect in planta protein behavior.…”
Correct subcellular targeting is crucial for protein function. Protein location can be visualized in vivo by fusion to a fluorescent protein (
FP
). Nevertheless, despite intense engineering efforts, most
FP
s are dim or completely quenched at low
pH
(<6). This is particularly problematic for the study of proteins targeted to acidic compartments such as vacuoles (
pH
~ 3–6) or plant cell walls (
pH
~ 3.5–8.3). Plant cell walls play important roles (e.g. structural/protective role, control of growth/morphogenesis), are diverse in structure and function, and are highly dynamic (e.g. during cell growth, in response to biotic/abiotic stresses). To study and engineer plant cell walls, it is therefore critical to identify robust tools which can be used to locate proteins expressed in the apoplast. Here we used a transient expression assay in
Nicotiana benthamiana
leaves to test a range of
FP
s in vivo
,
and determined which ones retained strong fluorescence in the acidic environment of the apoplast. We selected 10 fluorescent proteins with a range of in vitro properties; two historical
FP
s and eight
FP
s with in vitro properties suggesting lower
pH
sensitivity or improved brightness, some of which had never been tested in plants prior to our study. We targeted each
FP
to the cytosol or the apoplast and compared the fluorescence in both compartments, before testing the in vivo
pH
sensitivity of
FP
s across a
pH
8–4 gradient. Our results suggest that
mT
urquoise2,
mN
eonGreen, and
mC
herry are suited to tracking proteins in the apoplast under dynamic
pH
conditions. These fluorescent proteins may also be useful in other acidic compartments such as vacuoles.
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