FRET in donor-linker-acceptor constructs as detected by frequency-domain FLIM.

<p>The indicated constructs were expressed in HEK293 cells and FRET efficiency E was determined as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001916#s3" target="_blank">Material and Methods</a>. Shown are mean (bars), standard deviation (SD) and standard error of the mean (SEM) of 20–400 cells. For further detail, see text.</p

Summary of FRET changes of the constructs mentioned in this study.

<div><p>Mentioned are construct details (column 1–3), unique Lab ID (column 4), % ratio change (expressed as cAMP-induced change from an initial baseline ratio of 1, column 5) with its Standard Error of Mean, FRET efficiency E in rest (column 6) with SEM, E after saturation of the sensor with IBMX and Forskolin with SEM (column 7), absolute change of lifetime in ns with SEM (column 8) and % change in lifetime with SEM (column 9). Column 10 contains the fully descriptive name. As mentioned in the main text, we will from now on for brevity name the sensors "Epac-S^LabID", for example, Epac-S^H96 for mECFPΔ_Epac(CD, ΔDEP)_cp¹⁷³Ven_Ven.</p> <p>In gray is the reference construct Epac-S^H74. Abbreviations: nd, not determined; mCer3, monomeric Cerulean 3; mECFP, monomeric enhanced CFP; mTurq, monomeric Turquoise fluorescent protein; Hi-aff, High-affinity (Q270E mutant); ^cp173V, circular permutation of the fluorescent protein Venus(etc); td ^cp173V (etc), tandem of two ^cp173V fluorophores; ^cp173VV, tandem of cpVenus and Venus; CD, catalytically dead mutant (T781A & F782A amino acids of the Epac1 wild type protein); ΔDEP, deletion of the DEP domain to prevent membrane localization. Note that unlike Venus, Citrine contained the A206K monomerizing mutation. FRET efficiency E was calculated as E = 1-τ_{DonorAcceptor}/τ_Donor using τ_Donor = 4.10 ns for mTurquoise2 and 3.70 ns for mTurquoise1.</p></div

Characterization of novel FRET sensors.

<p>A. Localization of the FRET-sensors: N1E-115 cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)-^cp173Venus-Venus (Epac-S^H126); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td^cp173Venus (Epac-S^H187); mTurquoise2Δ-Epac(CD, ΔDEP)-td^cp173Dark Venus (Epac-S^H159) and mTurquoise2Δ-Epac(CD, ΔDEP)-^cp174Cit (Epac-S^H147). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S^H105); mTurquoise (Epac-S^H74) or mTurquoise2 (Epac-S^H126) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor ^cp173Venus-Venus (Epac-S^H134); td^cp173Venus (Epac-S^H187) or td^cp173Dark Venus (Epac-S^H189), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of HEK293T cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S^H126 or Epac-S^H134) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S^H187. After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S^H189. A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.</p

Schematic overview of the constructs used in this study.

<p>Donor and acceptor fluorophore are connected by a peptide stretch (Linker A: SGLRSRYPFASEL) or by the Epac1(ΔDEP, CD) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001916#pone.0001916-Ponsioen1" target="_blank">[1]</a>. Within this stretch, the amino acids PF were replaced by the Epac domain itself, leaving linkers B: SGLRSRY and C: ASEL. For truncated donor constructs (CFPΔ and GFPΔ) GITLGMDELYK was deleted from the donor FPs and SGLRS from the linker. In tandem acceptor constructs the acceptors were separated by a supplementary linker (Linker D: PNFVFLIGAAGILFVSGEL) except for tdHcRed and tdTomato which have distinctive linkers, namely NG(GA)₆PVAT) and (GHGTGSTGSGSSGTASSEDNNMA), respectively.</p

Recommended constructs for new experiments.

<p>Mentioned are construct details (column 1–3), Unique Lab ID (column 4), optimal application (Sensitized Emission, SE, or Fluorescence Lifetime IMaging, FLIM; column 5) and the fully descriptive name (column 6). Abreviations: GFP, Green Fluorescent Protein; mRFP, monomeric Red Fluorescent Protein; pAdeno, plasmid with Adenoviral backbone; pLVX, plasmid with LentiViral eXpression backbone; for other abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122513#pone.0122513.g002" target="_blank">Fig 2</a>. The lentiviral construct, H183) was prepared by dr. J. Karczewski (Wageningen University, NL) and has not been separately evaluated on our equipment.</p><p>Recommended constructs for new experiments.</p

UV-induced photochromism.

<p>Change in ratio of YFP to CFP emission in CFP^nd-linker-YFP^nd (squares) and CFP^nd-linker-Venus^d (triangles) following exposure to UV light for the indicated times. CFP^nd-linker-YFP^nd as well as free YFP (data not shown) display a dose-dependent increase in emission that maximizes at about 10%, whereas Venus and cp¹⁷³Venus (not shown) are insensitive to UV exposure. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001916#s3" target="_blank">Methods</a> for further detail.</p

Slow green-to-red maturation of tdTomato and its effects on FRET.

<p>(A) Cells expressing CFP^nd-EPAC-tdTomato for 24 hr display a spectrum of colors when viewed by eye using an Omega X154 triple-color (CFP-YFP-RFP) cube. For reproduction reasons, the confocal picture shows a mix of green (470–530 nm) and red (570–670) emission to closely match the image visible by eye. In contrast, CFP^nd-EPAC-mRFP and CFP^nd-EPAC-mCherry show a more homogeneous red color. (B) Cell-to-cell variability in maturation of CFP^nd-linker-tdTomato causes significant deviations in the fluorescence decay times detected in the CFP channel, as measured by frequency-domain FLIM. Scale bar, 12 µm.</p

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

<div><p>Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off (“blink”), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording.</p></div

pH in OxEA and Gloxy buffer.

<p>The pH in open dishes filled with 0.5 ml of OxEA (red squares) or Gloxy buffer (blue circles) is graphed at the indicated time points. Note the steep drop in pH in Gloxy buffer, which limits imaging to ~ 1 hour unless measures are taken to prevent oxygen influx.</p

Characterization of blinking in OxEA, Gloxy and MEA buffer.

<p>(A) Raw blinking frames (10 ms each, i.e. non-merged results) taken at the indicated time points. Shown are data for Alexa-488 (A488), Alexa-555 (A555) and Alexa-647 (A647) in both fresh Gloxy and OxEA buffer. Imaging was started after a 2–5 second pumping period at full laser power. Note presence of significant structured background in Gloxy buffer. (B) Mean intensity of individual blinks (merged in consecutive frames). Data are mean +/- SEM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#sec010" target="_blank">Methods</a> for further details. (C) Left panel, average number of blinks per frame (calculated in blocks of 1000 frames) of a preparation labeled with Alexa-488 and imaged in Gloxy or OxEA. Similar parts of cells with similar initial labeling density were selected based on the low-intensity wide-field image. Note the much larger number of blinks in OxEA for Alexa-488. Right panel, summary of blinks per frame data for Alexa-488, Alexa-555, Alexa-647 and FITC in three different buffers. Data are mean +/- SEM; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#sec010" target="_blank">Methods</a> for further details. (D) Number of blinks per frame, averaged over the full duration of the acquisition movie, in experiments carried out at the indicated times after applying the buffers. Within the hour, blinking has dropped dramatically in Gloxy whereas OxEA performs well for several hours. (E) Duration of individual blinks of Alexa-488 and Alexa-647 in fresh and ageing (90 minutes) Gloxy, and in fresh and ageing (120 min) OxEA. Multi-frame blinks are very common in ageing Gloxy buffer, as witnessed from the increased average duration of blinks and the enormous increase in duration spread (Data are mean +/- standard deviation). Increased blink duration adds to the appearance of structured background.</p