Symphotime 64

Manufactured by PicoQuant

Sourced in Germany

SymPhoTime 64 is a software package for data acquisition and analysis of time-resolved fluorescence experiments. It supports the collection and analysis of time-correlated single photon counting (TCSPC) data from PicoQuant instrumentation.

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Lab products found in correlation

Ldh d c 485, by PicoQuant (10 mentions) Microtime 200, by PicoQuant (8 mentions) Picoharp 300, by PicoQuant (5 mentions) Time correlated single photon counting module, by PicoQuant (4 mentions) Eclipse ti a1r microscope, by PicoQuant (3 mentions) Timeharp 260 pico board, by PicoQuant (3 mentions) Hydraharp 400 tcspc module, by PicoQuant (3 mentions) Microtime 200 microscope, by PicoQuant (3 mentions) Fv1200 confocal microscope, by Olympus (3 mentions) Pma hybrid 40, by PicoQuant (3 mentions) Hydraharp 400, by PicoQuant (3 mentions) Micro incubator, by Okolab (2 mentions) Plastic chambers, by Ibidi (2 mentions) Lsm980 multiphoton microscope, by PicoQuant (2 mentions) Tcspc flim module, by PicoQuant (2 mentions) Timeharp 260 card, by PicoQuant (2 mentions) Dmi8 inverted microscope, by Leica (2 mentions) Picoharp 300 tcspc module, by PicoQuant (2 mentions) Tcs sp8 smd, by Leica (2 mentions) Micro time 200, by Olympus (1 mentions)

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SymPhoTime (22 citations)

90 protocols using symphotime 64

Fluorescence Correlation Spectroscopy of RNCs

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For FCS measurements, the RNCs were diluted to 5–7 nM in HiFi buffer. Measurements were performed using a confocal microscope FCS set‐up (Micro Time 200, Olympus IX73) (PicoQuant, Berlin, Germany) at 636.5 nm wavelength continuous wave laser with power ~40 μW and 1–1.5 molecules per observation volume (Liutkute et al, 2020a (link)). Data processing and autocorrelation function (ACF) calculations were done using SymPhoTime 64 software (PicoQuant, Berlin, Germany). Each sample was subject to 4 measurements of 10 min, which were averaged. RNCs of each CspA variant were independently prepared and measured 2 times, generating an overall of 8 technical replicates for each final ACF.
Fitting of initial ACFs was carried out using a model (Liutkute et al, 2020a (link)) for single species diffusion with two relaxation rate constants, a triplet rate constant, and a diffusion rate constant,

G (τ) = (1 + c_{1} e^{‐ k_{1} t} + c_{2} e^{‐ k_{2} t}) (\frac{1 ‐ F + F e^{‐ k_{f} t}}{1 ‐ F}) (\frac{1}{N}) {(1 + k_{d} t)}^{‐ 1}

where k₁ and k₂ are apparent relaxation rate constants with respective amplitudes c₁ and c₂, N is the average number of molecules in the confocal volume, F is the amplitude for the triplet component with rate constant k_f, and k_d is the inverse diffusion time. All curves were fitted independently of each other and results are displayed in Appendix Table S4.

Agirrezabala X., Samatova E., Macher M., Liutkute M., Maiti M., Gil‐Carton D., Novacek J., Valle M, & Rodnina M.V. (2022). A switch from α‐helical to β‐strand conformation during co‐translational protein folding. The EMBO Journal, 41(4), e109175.

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Cellular Imaging of O-GlcNAcylation

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The cellular imaging of O-GlcNAcylation on a specific protein was described previously^{38 (link)}, and adapted for this work. Briefly, after the transfection of EGFP-ESRRB and 1 mM GalNAz treatment for 48 h, the HeLa cells were washed, fixed and permeabilized, followed by click-labeling used for FLIM-FRET imaging. FLIM-FRET imaging was performed on a TCS SP8X scanning confocal microscope (Leica). Images and distribution histograms of fluorescence lifetime were acquired by TCSPC software (SymPhoTime 64 software, PicoQuant GmbH).

Hao Y., Fan X., Shi Y., Zhang C., Sun D.E., Qin K., Qin W., Zhou W, & Chen X. (2019). Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells. Nature Communications, 10, 4065.

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FLIM Imaging of Membrane Probes

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FLIM imaging was performed using a Nikon Eclipse Ti A1R microscope equipped with a Time Correlated Single-Photon Counting module from PicoQuant59 . Excitation was performed using a pulsed 485nm laser (PicoQuant, LDH-D-C-485) operating at 20 MHz, and emission signal was collected through a bandpass 600/50nm filter using a gated PMA hybrid 40 detector and a TimeHarp 260 PICO board (PicoQuant). SymPhoTime 64 software (PicoQuant) was then used to fit fluorescence decay data (from full images or regions of interest) to a dual exponential model after deconvolution for the instrument response function (measured using the backscattered emission light of a 1µM fluorescein solution with 4M KI). Data was expressed as means ± standard deviation of the mean. The full width at half-maximum (FWHM) response of the instrument was measured at 176 ps.
Supplementary figure 1a shows all parameters extracted from the fits of FliptR in various GUV compositions. As similar tendencies were seen for τ₁and τ₂, the longest lifetime (τ₁) obtained by the double exponential fits was used for all subsequent graphs.

Colom A., Derivery E., Soleimanpour S., Tomba C., Dal Molin M., Sakai N., González-Gaitán M., Matile S, & Roux A. (2018). A Fluorescent Membrane Tension Probe. Nature chemistry, 10(11), 1118-1125.

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Fluorescence Lifetime Imaging Microscopy

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FLIM measurements were performed on an Olympus FluoView FV-1000 system (Olympus, Tokyo, Japan) equipped with a time-resolved LSM upgrade (PicoQuant GmbH, Berlin, Germany) using a 60X, 1.2NA water immersion objective. Images of 512 × 512 pixels per frame were acquired after excitation with a pulsed-laser diode at 488 nm. Fluorescence was detected using a SPAD detector and a 520/35 nm bandpass filter. In each measurement, a minimum of 10⁵ photons were recorded by accumulation of 60 frames over a time period of 90 s. Regions of interest in the cytoplasm of cells were analysed using SymPhoTime64 software (PicoQuant GmbH, Berlin, Germany) taking into account the instrument response function determined by measuring a saturated Erythrosine B solution according to manufacturer’s instructions. Resulting decay curves were fitted using a mono-exponential function.

Dunsing V., Luckner M., Zühlke B., Petazzi R.A., Herrmann A, & Chiantia S. (2018). Optimal fluorescent protein tags for quantifying protein oligomerization in living cells. Scientific Reports, 8, 10634.

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Fluorescence Lifetime Imaging of M4 Peptides and PIP2

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Fluorescence lifetimes were evaluated at room temperature in a drop placed on a coverslip and inserted in an inverted confocal microscope IX83 (Olympus, Hamburg, Germany) equipped with TimeHarp 260 PICO time-correlated single-photon counting electronics and cooled GaAsP hybrid detectors (all PicoQuant, Berlin, Germany). The M4 peptides or PIP₂ fluorescence were excited at 485 nm by an LDH-485 picosecond laser head (PicoQuant, Berlin, Germany). Emission decays were collected in the epifluorescence mode using a combination of a 488-nm dichroic reflector (Olympus, Hamburg, Germany) and a Semrock 520/35 bandpass filter in the detection path. Fluorescence was assumed to decay multiexponentially according to the formula:

I (t) = \sum_{i} α_{i} \times e x p^{(\frac{- t}{τ_{i}})}

where τ_i are fluorescence lifetimes and α_i the corresponding amplitudes. The intensity-weighted mean fluorescence lifetime was calculated as:

τ_{m e a n} = \sum_{i} α_{i} \times τ_{i}^{2} / \sum_{i} α_{i} \times τ_{i}

The least-squares deconvolution fitting was performed by the SymPho Time 64 software (PicoQuant).

Bousova K., Barvik I., Herman P., Hofbauerová K., Monincova L., Majer P., Zouharova M., Vetyskova V., Postulkova K, & Vondrasek J. (2020). Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4. International Journal of Molecular Sciences, 21(12), 4323.

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FRET-FLIM Analysis of PtoMYB221-PtoUBC34s Interaction

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The interaction between PtoMYB221 and PtoUBC34s was described in a previous study [47 (link)]. The donor vector pGreen0029-35S:YFP-PtoUBC34s and receptor vector pGreen0029-35S:PtoMYB221-RFP (Fig. S6h) were co-transferred into poplar leaves via Agrobacterium-mediated syringe infiltration as described above. FRET-FLIM was performed on an Olympus inverted FV1200 microscope additionally equipped with a Picoquant picoHarp300 (Germany) controller according to the reported method [74 (link)]. The YFP-PdbUBC34s was excited at 488 nm using a picosecond pulsed diode laser operated at a repetition rate of 40 MHz through an objective (40× water immersion, NA 1.2). The emitted light was collected in the same objective and filtered with a 520/35 nm bandpass filter. Fluorescence was then detected by an MPD SPAD detector. The region of interest in the images was selected and acquired with acquisition photons of up to 20,000 or more. SymphoTime 64 software (PicoQuant, Germany) was used to calculate the decay curves per pixel and fitted with a decay model. Double-exponential was selected for the test combination with donor YFP-PdbUBC34s and receptor PdbMYB221-RFP, and the mono-exponential model was applied for only donor YFP-PdbUBC34s as a control.

Zheng L., Yang J., Chen Y., Ding L., Wei J, & Wang H. (2021). An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar. BMC Plant Biology, 21, 54.

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Measuring Adipocyte Membrane Tension in Mice

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Primary visceral adipocytes from mice fed with standard diet or HFD for 7 days were isolated and incubated with 1 μM of the membrane tension probe Flipper-TR® (Tebu-bio, #SC020) for 30 min at 37 °C as described^{29 (link)}. The adipocytes were then washed 3 times with HBSS and imaged with a Leica-SP8 FLIM microscope. Excitation was performed using a pulsed 488 nm laser (Laser kit WLL2+pulse picker, Leica Microsystems) operating at 80 MHz, and the emission signal was collected from 549 to 651 nm with acousto-optical beam splitter (AOBS) using a gated hybrid (HyD SMD) detectors and a TimeHarp 300 TCSPC Module and Picosecond Event Timer (PicoQuant). SymPhoTime 64 software (PicoQuant) was then used to fit fluorescence decay data. To extract lifetime information, the photon histograms from membrane regions were fitted with a double exponential, and 2 fluorescence emission decay times (τ1 and τ2) are extracted. The longest lifetime with the higher fit amplitude τ1 is used to report membrane tension^{29 (link)}.

Wang S., Cao S., Arhatte M., Li D., Shi Y., Kurz S., Hu J., Wang L., Shao J., Atzberger A., Wang Z., Wang C., Zang W., Fleming I., Wettschureck N., Honoré E, & Offermanns S. (2020). Adipocyte Piezo1 mediates obesogenic adipogenesis through the FGF1/FGFR1 signaling pathway in mice. Nature Communications, 11, 2303.

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Circularly Polarized Pulsed Laser Setup

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A 530-nm circularly polarized pulsed laser light (PicoQuant, LDH-P-FA-530L; 120 W cm⁻² at the sample plane) was used as the excitation source. Hanbury–Brown and Twiss-type photon correlation configurations were applied. The fluorescence photons of all wavelengths were divided by a 50/50 non-polarizing cube beam splitter (Thorlabs, CM1-BS013) and detected by two SPADs (PicoQuant, τ-SPAD-50,) after they pass through a set of bandpass filters (Semrock, FF01-665/150). The signals from the two SPADs were recorded by the TCSPC HydraHarp 400 module in the time-tagged time-resolved mode. SymPhoTime64 software (PicoQuant) was used for both the acquisition of all fluorescence microscopy data and analysis of the data.

Piwoński H., Michinobu T, & Habuchi S. (2017). Controlling photophysical properties of ultrasmall conjugated polymer nanoparticles through polymer chain packing. Nature Communications, 8, 15256.

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Fluorescence Lifetime Imaging of Receptor Aggregation

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Fluorescence Lifetime images were obtained using a Leica DMi8 inverted microscope and a PicoHarp 300 time correlated single photon counting (TCSPC) module plus picosecond event timer (PicoQuant GMBH Berlin, Germany #973001). A Leica Harmonic Compound PL apochromatic CS2 63X water objective with a correction collar (1.2 NA) was used for imaging. Cells were imaged at 35°C with temperature maintained using a Bioptics Objective heater. A tunable White Light Laser (470 – 670 nm) with 4% laser power and a 488 nm notch filter (donor excitation) and 561 nm notch filter (acceptor excitation) was used to excite samples with a pulse rate of 80 mHz. A scan speed of 700 and a 256×256 resolution was used. Two hybrid detectors collected photons at (498–532 nm) and (608–650 nm) respectively on the photon counting mode setting, with only the 488 notch filter active and the 498–532 hybrid detector active during FLIM acquisition. Fluorescence decay rate was measured over 20–25 laser pulse repetitions and the data was fit to either one or two component reverse exponential equations using Symphotime 64 software (PicoQuant) to calculate average lifetime. An intensity-based threshold was employed to discern between receptors in aggregates and receptors outside aggregates. Imaging was performed at the basal membrane.

Rinaldi D.A., Kanagy W.K., Kaye H.C., Grattan R.M., Lucero S.R., Pérez M.P., Wester M.J., Lidke K.A., Wilson B.S, & Lidke D.S. (2023). Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcεRI Aggregation and Structural Changes. bioRxiv.

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Statistical Analysis of Fluorescence Intensity Decays

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Statistical significance was determined by two-tailed Student’s t test using PRISM software (5.00, GraphPad Software; Figs. 1, 2, 5, and S2; and Tables S1 and S2). The resulting p-values are indicated as follows: n.s., P > 0.05; *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; and ***, P < 0.001. To judge the goodness of one- and two-exponential theoretical fits of fluorescent intensity decays in FLIM-FRET, χ² analyses were done using Symphotime 64 software (Picoquant; Tables S3 and S4). To compare the fits, the extra sum-of-squares F test was applied using SPSS software (version 19; IBM SPSS for Windows; Tables S3, S4, S5, and S6). For parametric tests, data distribution was assumed to be normal but was not formally tested.

Wang S., Wu C., Zhang Y., Zhong Q., Sun H., Cao W., Ge G., Li G., Zhang X.F, & Chen J. (2018). Integrin α4β7 switches its ligand specificity via distinct conformer-specific activation. The Journal of Cell Biology, 217(8), 2799-2812.

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