Lifetime measurements were carried out on a confocal laser-scanning microscope (FluoView 1000, Olympus) equipped with a FLIM/FCS upgrade kit from PicoQuant using a 60× (NA 1.2) water-immersion objective (UPLSAPO, Olympus). EGFP/mNeonGreen was excited either by the 488 nm line of an argon laser (Olympus) for cLSM/FRAP or by a picosecond pulsed 485 nm laser diode at 40 MHz repetition rate (LDH-D-C-485, Picoquant). Time-correlated single photon counting (TCSPC) was performed using the TCSPC module PicoHarp 300 (PicoQuant together with a picosecond laser driver Sepia II (PicoQuant) and a single photon avalanche detector (PicoQuant)). Emission photons were filtered by a 500–550 nm bandpass filter (BrightLine HC 525/50, Semrock). TCSPC was acquired using point measurement or image mode (i.e. FLIM mode). Acquisition time was more than 90 s to obtain >105 counts per sample for robust lifetime analyses. If not mentioned elsewhere, the TCSPC histograms were tail-fit with mono-exponential decay function using SymPhoTime64 integrated in the PicoQuant system. Only for mNeon-Ypt7 on graphene, the TCSPC histograms were fit with bi-exponential decay functions by fixing one component to the obtained average lifetime of mNeon-Ypt7 on glass (<15%).
Sepia 2
Manufactured by PicoQuant
Sourced in Japan, Germany
The Sepia II is a time-correlated single photon counting (TCSPC) system developed by PicoQuant. It is designed for time-resolved fluorescence measurements, providing high-resolution timing capabilities. The Sepia II offers advanced functionality for fluorescence lifetime imaging microscopy (FLIM) and other time-resolved spectroscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Picoharp 300, by PicoQuant (2 mentions)
Fluoview 1000, by Olympus (2 mentions)
Ldh d c 485, by PicoQuant (1 mentions)
Uplsapo, by Olympus (1 mentions)
Pdl 828, by PicoQuant (1 mentions)
Microtime 200, by PicoQuant (1 mentions)
Igor pro version 6.22a, by Wavemetrics (1 mentions)
Ldh d c 640, by PicoQuant (1 mentions)
Uplansapo, by Olympus (1 mentions)
Spcm cd3516h, by Excelitas (1 mentions)
Tcspc system, by PicoQuant (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Picoharp 300 tcspc module, by PicoQuant (1 mentions)
Symphotime software, by PicoQuant (1 mentions)
6 protocols using sepia 2
1
Fluorescence Lifetime Microscopy on Graphene
2
Time-Resolved Photoluminescence Measurements
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Time-resolved photoluminescence measurements
were carried out using
a confocal microscope setup (PicoQuant, MicroTime 200). A 405 nm pulsed
diode laser (PDL 828 “SEPIA II”, PicoQuant, pulse width
∼100 ps) was focused onto the surface of the microcrystalline
powder sandwiched between two glass coverslips using an air objective
(20 ×, 0.4 NA). The emission signal was separated from the excitation
light using a dichroic mirror (Z405RDC, Chroma). Repetition rates
of 0.2 MHz were used with an average energy density of 2.5 μJ/cm2. Photoluminescence photon arrival times were binned in 400
ps intervals.
were carried out using
a confocal microscope setup (PicoQuant, MicroTime 200). A 405 nm pulsed
diode laser (PDL 828 “SEPIA II”, PicoQuant, pulse width
∼100 ps) was focused onto the surface of the microcrystalline
powder sandwiched between two glass coverslips using an air objective
(20 ×, 0.4 NA). The emission signal was separated from the excitation
light using a dichroic mirror (Z405RDC, Chroma). Repetition rates
of 0.2 MHz were used with an average energy density of 2.5 μJ/cm2. Photoluminescence photon arrival times were binned in 400
ps intervals.
3
FLIM Analysis of Protein-Protein Interactions
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FLIM experiments were performed on an Olympus FluoView 1000 laser scanning microscope (Olympus, Tokyo, Japan) with a Sepia II and PicoHarp 300 time correlated single photon counting system from PicoQuant (Berlin, Germany). Proteins were tagged with the fluorescent proteins mCitrine (donor) and mCherry (acceptor) to optimize FRET efficiency. All experiments were carried out at 37°C with a 60x water objective. For imaging, samples were simultaneously excited with 488 (10% laser intensity) and 561 nm (31% laser intensity) light using a 405/488/561/633 dichroic mirror. Excitation light was split with a dichroic mirror at 560 nm and detected between 500–550 nm (green channel) and 580–680 nm (red channel). The pinhole was set to 300 μm. FLIM images were acquired using 470 nm excitation (36% intensity) with a pulse frequency of 40 MHz, a 405/470 dichroic mirror, and a 525/15 band path filter. FLIM images were analyzed using IGOR Pro (Version 6.22 A, Wave Metrics, Lake Oswego, OR) with pFLIM3 as previously described (Walther et al., 2011 (link)).
4
Dual-Laser Confocal Fluorescence Fluctuation Spectroscopy
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Measurements were performed on an Olympus IX73 inverted microscope stand equipped with a 1.2 N.A. water-immersion 60× superapochromat objective (UplanSApo; Olympus) and suitable emission and excitation bandpass filters (Semrock and AHF). Two pulsed diode lasers (LDH-P-FA-530 and LDH-D-C-640; PicoQuant) were operated at 40 MHz for pulse interleaved excitation dcFLCCS (Sepia II; PicoQuant). Emitted photons were detected in two separated channels coupled with two SPAD detectors (SPCM CD3516H; Excelitas) and a time-correlated single-photon counting unit to generate picosecond histograms also called lifetime spectra (16-ps resolution; HydraHarp 400) from the statistical photon arrival times. The laser powers were set to 20 µW for the LDH-P-FA-530 and to 17 µW for the LDH-D-C-640 laser and the intensity fluctuation recorded for 120 s with a correlation integration time taken as 2 s. The confocal volume was calibrated using free dyes of known diffusion constants D (using Rhodamine B in excitation channel 530 with D = 426.4 µm2/s at 298 K and a structural parameter of S = 4, and Atto-655NHS ester in excitation channel 640 with D = 403.6 µm2/s at 298 K and a structural parameter of S = 4). All measurements were performed 20 µm away from the coverslip.
5
Time-resolved Fluorescence Lifetime Imaging
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Time domain FLIM experiments were performed on a Time Correlated Single Photon Counting (TCSPC) system (PicoQuant) attached to an Olympus FV-1000 confocal microscope (Olympus) with a 60 × 1.2 W objective. The excitation light source was a 485-nm pulsed diode laser controlled by a Sepia II (PicoQuant) driver with a dichroic mirror of 488/559 and a 520/30 emission filter. Individual photon arrivals were detected using a SPAD detector, and events were recorded by a PicoHarp 300 TCSPC module. Lifetime analysis was carried out using SymPhoTime 200 software. Mono- and bi-exponential fittings were applied. The percentage of binding was calculated from the amplitudes derived from the bi-exponential fitting, as previously shown (Orthaus et al., 2009 ).
6
FLIM-FRET Analysis of RABE1b-CFP and ANN5-YFP
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For FLIM-FRET (Fluorescence Lifetime Imaging Microscopy-Förster Resonance Energy Transfer) RABE1b was fused to CFP (donor) and transiently expressed in N. benthamiana leaves in the presence or absence of the potential interacting partner ANN5 fused to YFP (acceptor). Cells were imaged with an FV100 confocal system (Olympus, Tokyo, Japan) equipped with a 60× water immersion objective lens. For FLIM CFP fusion protein was excited with a 440 nm pulsed diode laser (Sepia II, PicoQuant, Berlin, Germany) and detected using a 482/35 bandpass filter. Images were acquired with a frame size of 256 × 256 pixels. Photons were collected with a SPAD detector and counted with the PicoHarp 300 TCSPC module (Picoquant). The obtained data were analyzed with Symphotime software (PicoQuant). Fluorescence lifetimes of CFP in plastid nucleoids were calculated by fitting a bi-exponential decay model.
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