The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Germany) and a ~ 70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Japan). The fluorescent signal from o-BMVC was collected using a 60 × NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Japan), passed through a 550/88 nm bandpass filter (Semrock, USA), and detected using a single-photon avalanche diode (SPAD) (PD-100-CTC; Micro Photon Devices, Italy). The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software (PicoHarp 300 and SymPhoTime v5.3.2; PicoQuant, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.
Symphotime v5
Manufactured by PicoQuant
Sourced in Germany
SymPhoTime v5.3.2 is a comprehensive software package for time-resolved data acquisition and analysis. It supports a wide range of time-resolved techniques, including time-correlated single-photon counting (TCSPC), fluorescence lifetime imaging (FLIM), and photon timing experiments. The software provides tools for data visualization, analysis, and fit modeling.
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7 protocols using symphotime v5
1
Picosecond Laser FLIM Imaging of o-BMVC
2
Quantitative FLIM Imaging of DNA Structures
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The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Germany) and a ~70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Japan). The fluorescent signal from of o-BMVC was collected using a 60× NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Japan), passing through a 550/88 nm bandpass filter (Semrock, USA), followed by detection using a SPAD (PD-100-CTC; Micro Photon Devices, Italy). The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software (PicoHarp 300 and SymPhoTime v5.3.2; PicoQuant, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.
For the study of fixed cells, cells on coverslip were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of PDS pretreatment, HeLa and MRC-5 cells on coverslip were treated with 1 µM PDS overnight. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of DNase treatment, HeLa cells on coverslips were fixed with 70% ethanol for 10 min and then treated with 20 µg/ml DNase for 1 h at 37 °C followed by 5 µM o-BMVC staining for 10 min.
For the study of fixed cells, cells on coverslip were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of PDS pretreatment, HeLa and MRC-5 cells on coverslip were treated with 1 µM PDS overnight. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of DNase treatment, HeLa cells on coverslips were fixed with 70% ethanol for 10 min and then treated with 20 µg/ml DNase for 1 h at 37 °C followed by 5 µM o-BMVC staining for 10 min.
3
Fluorescence Lifetime Imaging of G4 Ligands
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The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Berlin, Germany) and a ~70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Tokyo, Japan). The fluorescent signal from o-BMVC was collected using a 60× NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Japan), passing through a 550/88 nm bandpass filter (Semrock, Rochester, NY, USA), followed by detection using a SPAD (PD-100-CTC; Micro Photon Devices, Bolzano, Italy). The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software (PicoHarp 300 and SymPhoTime v5.3.2; PicoQuant, Berlin, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.
For the study of G4 ligands pretreatment, 10 µM TMPyP4, BMVC4 and BRACO-19 were used for pretreatment of HeLa cells and H33258 for pretreatment of MRC-5 cells. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. Quantitative analysis of o-BMVC foci by using the Otsu algorithm for the image analysis was described previously [10 (link)].
For the study of G4 ligands pretreatment, 10 µM TMPyP4, BMVC4 and BRACO-19 were used for pretreatment of HeLa cells and H33258 for pretreatment of MRC-5 cells. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. Quantitative analysis of o-BMVC foci by using the Otsu algorithm for the image analysis was described previously [10 (link)].
4
Fluorescence Lifetime Imaging Microscopy of o-BMVC
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The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Berlin, Germany) and a ~70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Tokyo, Japan). The fluorescent signal from the o-BMVC was collected using a 60 × NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Tokyo, Japan) passing through a 550/88 nm bandpass filter (Semrock, Rochester, New York, NY, USA), followed by detection using a SPAD (PD-100-CTC; Micro Photon Devices, Bolzano, Italy), the time resolution of which was less than 50 ps FWHM. The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software by mono-exponential curve fitting (PicoHarp 300; electrical time resolution was less than 25 ps; and SymPhoTime v5.3.2; PicoQuant, Berlin, Germany). FLIM images were constructed from pixel-by-pixel lifetime information. Time-gated FLIM images had set time windows at 2.4 ns to separate the image into two colors: white (decay time ≥ 2.4 ns) and red (decay time < 2.4 ns).
5
Fluorescence Lifetime Imaging of o-BMVC
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The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Berlin, Germany) and a ~70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Tokyo, Japan). The fluorescent signal from the o-BMVC was collected using a 60 × NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Tokyo, Japan) passing through a 550/88 nm bandpass filter (Semrock, Rochester, New York, NY, USA), followed by detection using a SPAD (PD-100-CTC; Micro Photon Devices, Bolzano, Italy), the time resolution of which was was less then 50 ps FWHM. The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software by mono-exponential curve fitting (PicoHarp 300, electrical time resolution was less then 25 ps and SymPhoTime v5.3.2; PicoQuant, Berlin, Germany). FLIM images were constructed from pixel-by-pixel lifetime information. Time-gated FLIM images had set time windows at 2.4 ns to separate the image into two colors: white (decay time ≥ 2.4 ns) and red (decay time < 2.4 ns). Live CL1-0 cells were incubated with 5 µM o-BMVC and its complexes with 15 µM GROs for 2 h. For the unfolded study, samples were washed twice with phosphate-buffered saline (PBS) followed by the addition of 15 µM anti-CMA.
6
Fluorescence Lifetime Imaging Analysis
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FLIM analysis was carried out using SymPhoTime v5.4.4 (PicoQuant). The measured fluorescence decay was fitted to a bi-exponential decay model using the maximum-likelihood estimation method. Fluorescence lifetime values were determined by pixel-by-pixel fitting, which was guided by an initial global decay fit. Reported fluorescence lifetimes are an amplitude-weighted average of the two decay components, тamp= (A1т1 + A2т2) / (A1 + A2) (where A1 and A2 are the amplitudes of the decay components and т1 and т2 are the fluorescence lifetimes) and were only calculated for pixels with greater than 300 photon counts. FRET efficiency, EFRET = 1 – тDA/тD, where τDA is the lifetime of the donor (GFP) in the presence of the acceptor (mCherry) and тD is the single pixel median amplitude-weighted fluorescence lifetime reported for the GFP donor-only sample, calculated per pixel from the GFP-mCherry samples. Image maps representing FRET efficiency per pixel were constructed in R using the spatstat package.
7
Anomalous Diffusion Analysis of Neuronal Proteins
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Autocorrelation traces were generated from the photon-counting histograms for each 5 to 30 s measurement using SymPhoTime v5.4.4 software (Picoquant, Germany). In vitro calibration traces were fitted using the Triplet model (three-dimensional free diffusion model with triplet state) with informed diffusion values to yield Veff and κ values. Neuronal autocorrelation traces were fitted using a Triplet Extended model (two-dimensional anomalous diffusion model with triplet state), this model is designed for fluorescent molecules moving within a plane, for example, proteins in a membrane. Diffusion within the cells is expected to be anomalous; therefore, the anomaly parameter was not fixed to one. The anomaly parameter (α) measures the departure from free Brownian diffusion (α=1) to either superdiffusion (α>1) or subdiffusion (α<1) for a diffusing species. Autocorrelation curves with (α>1) display the sharpest decay, whereas the those with α<1 decrease quite slowly55 (link).
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