Fig 1.
Spectrum of mNeonGreen, mRuby3, mScarlet-I and mCherry.
Absorbance (dashed lines) and emission spectrum (solid lines) of purified mNeonGreen (green lines) overlaid with the spectrum of purified mRuby3 (A), mScarlet-I (B), and mCherry (C). Spectrum were obtained from the reference indicated in Table 1.
Table 1.
Properties of the fluorescent proteins used in this study.
Fig 2.
Spectral FRET of each mNeonGreen-Red FP tandem constructs.
(A) Cartoon schematic of the mNeonGreen-Red FP tandem constructs used for FRET experiments. (B) Average emission scan of cells expressing NG-Stop (black) when excited at 470 nm overlaid with the reported spectrum for purified mNeonGreen in green (n = 3 independent transfections). Example raw emission spectrum (pink) of tandem (C) NG-mRuby3, (D) NG-mScarlet-I, and (E) NG-mCherry when excited at 470 nm. The dashed black line shows the sum of donor (green) and acceptor (red) components calculated by linear unmixing. (F) FRET efficiencies calculated from the spectrum for each construct (n = 3). *** = P < 0.0005 between the indicated conditions.
Fig 3.
mNeonGreen performs well under various laser powers for two-photon time domain FLIM acquisitions.
(A) Lifetime data collected from individual HEK293 cells expressing cytosolic mNeonGreen at various laser powers up to 25 W/cm2 after 50 frames. Black bars indicate the average ± 95% confidence interval. (B) Lifetime and (C) intensity of samples taken over 300 frames at various laser powers. * = P < 0.05, ** = P< 0.005, and *** = P < 0.0005 compared to the frame matched 5W/cm2 dataset. N for each sample is as follows 5W/cm2: 11 cells, 10W/cm2: 10 cells, 15W/cm2: 14 cells, 20W/cm2: 9 cells, 25W/cm2: 11 cells. (D) Example lifetime decay curves obtained at 15 W/cm2 over 300 frames. (E) Example lifetime decay and normalized decays (F) obtained at 25 W/cm2 over 300 frames.
Fig 4.
Lifetime of mNeonGreen-Red FP tandem constructs.
(A) The lifetime of mNeonGreen in individual cells expressing a mNeonGreen-Red Protein tandem fusion construct. Black bars indicate the average ± 95% confidence interval. (B) FRET efficiency calculations for each tandem construct. *** = P < 0.0005 compared to NG-Stop and # indicates P < 0.0005 compared to NG-Stop and P < 0.0005 compared to NG-mRuby3. NG-mScarlet-I and NG-mCherry are not statistically different (P = 0.75) (C) Example decay curves for each tandem representative of the average lifetime of all cells for each construct. (D) Example lifetime heat maps for a single frame for each construct. N for each construct is as follows NG-Stop: 68 cells, NG-mRuby3: 63 cells, NG-mScarlet-I: 64 cells, and NG-mCherry: 64 cells.
Fig 5.
Confocal microscopy of the tandem constructs.
(A) Example images of NG-Red FP constructs when directly excited by 499 nm or 559 nm lasers. Histograms of the slopes of the red/green intensity correlations for individual cells expressing (B) NG-mRuby3 (n = 1077 cells), (C) NG-mScarlet-I (n = 1745 cells), and (D) NG-mCherry (n = 1557 cells).
Fig 6.
Behavior of distinct mRuby3 and mScarlet containing tandem constructs.
(A) The lifetimes of a distinct set of NG-Stop express cells with the lifetimes of 2 new mRuby3 containing tandem constructs and an mScarlet containing construct. Black bars indicate the average ± 95% confidence interval. *** = P < 0.0005 compared to NG-Stop. NG-Stop and NG-P2A-mRuby3 are not statistically different (P = 0.65). N for the lifetime measurements of each construct is as follows NG-Stop: 64 cells, mRuby3-GGSGG-NG: 66 cells, NG-P2A-mRuby3: 69 cells, and NG-mScarlet: 77 cells. (B) Average FRET efficiency for each of the new tandem constructs. (C) Example merge confocal images of HEK293 cells expressing each of the new tandem constructs. Pooled histograms of the slopes of cells expressing (D) mRuby3-GGSGG-NG, (E) NG-P2A-mRuby3, and (F) NG-mScarlet. N for the confocal measurements of each construct is as follows mRuby3-GGSGG-NG: 2302 cells, NG-P2A-mRuby3: 1548 cells, and NG-mScarlet: 1150 cells.
Fig 7.
FRET of NG-mRuby3 1–5 days post transfection.
(A) Lifetimes of cells expressing NG-mRuby3 construct 1–5 days post transfection (DPT) with 1 DPT is replicated from Fig 3A for reference. Black bars indicated the average lifetime ± 95% confidence interval. The green shading indicates the range of lifetimes observed from the NG-Stop construct. *** = P < 0.0005 compared to 1 DPT, and # = P < 0.0005 compared to 1 DPT and P < 0.0005 compared to the day before. N for each condition is as follows, 2 DPT: 30 cells, 3 DPT: 34 cells, 4 DPT: 35 cells, and 5 DPT: 31 cells. (B) Average FRET efficiency of NG-mRuby3 1–5 DPT. (C) Example lifetime maps collected each day tested. (D) Lifetime data from NG-mRuby3 expressing cells 5 days post transfection before and after acceptor photobleaching (n = 15). *** = P < 0.0005 after photobleaching compared to before photobleaching. (E) Example lifetime maps of the same cells before and after acceptor photobleaching.