Single-molecule ALEX experiments were performed at room temperature (22°C) using a home-built confocal microscope similar to the described setup (Gouridis et al., 2015 (link)). In brief, two laser-diodes (Obis, Coherent, USA) with emission wavelength of 532 and 637 nm were directly modulated for alternating periods of 50 μs and used for confocal excitation. The laser beams where coupled into a single-mode fiber (PM-S405-XP, Thorlabs, UK) and collimated (MB06, Q-Optics/Linos) before entering a water immersion objective (60X, NA 1.2, UPlanSAPO 60XO, Olympus). The fluorescence was collected by excitation at a depth of 20 μm. Average laser powers were 30 μW at 532 nm (∼30 kW/cm2) and 15 μW at 637 nm (∼15 kW/cm2). Excitation and emission were separated by a dichroic beam splitter (zt532/642rpc, AHF Analysentechnik), mounted in an inverse microscope body (IX71, Olympus). Emitted light was focused onto a 50 μm pinhole and spectrally separated (640DCXR, AHF Analysentechnik) on two APDs (τ-spad, < 50 dark-counts/s, Picoquant) with appropriate spectral filtering (donor channel: HC582/75; acceptor channel: Edge Basic 647LP; both AHF Analysentechnik). The signal was recorded using custom-written LabVIEW software (Kapanidis et al., 2004 (link)).
τ spad
Manufactured by PicoQuant
Sourced in Germany
The τ-SPAD is a high-performance single-photon avalanche diode (SPAD) detector developed by PicoQuant. It is designed for time-resolved fluorescence and photon correlation spectroscopy applications. The τ-SPAD offers high photon detection efficiency, low timing jitter, and excellent timing resolution, making it a versatile tool for various scientific and industrial research applications.
Automatically generated - may contain errors
Lab products found in correlation
Picoharp 300, by PicoQuant (3 mentions)
Dpc 230 photon correlator, by Becker & Hickl (2 mentions)
τ spad, by Becker & Hickl (2 mentions)
Pylon, by Teledyne (1 mentions)
Acton sp2500, by Teledyne (1 mentions)
Uplsapo 60xo, by Olympus (1 mentions)
Pln20x, by Olympus (1 mentions)
Uplsapo 60, by Olympus (1 mentions)
Microtime 200, by PicoQuant (1 mentions)
Mdl 300, by PicoQuant (1 mentions)
Pixis 1024b, by Teledyne (1 mentions)
Hydraharp 400, by PicoQuant (1 mentions)
Ldh p fa 530l, by PicoQuant (1 mentions)
11 protocols using τ spad
1
Single-Molecule ALEX Microscopy Setup
2
Cryogenic Nanophotonics Characterization Setup
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PL experiments were performed in a lab-built cryogenic setup. The sample was mounted on piezo-stepping units (attocube systems ANPxy101 and ANPz102) for positioning with respect to a low-temperature objective (attocube systems LT-APO/NIR/0.81) or the cavity mode. The microscope was placed in a dewar with an inert helium atmosphere at a pressure of 20 mbar and immersed in liquid helium at 4.2 K. Excitation around 635–705 nm was performed with a wavelength-tunable supercontinuum laser (NKT SuperK Extreme and SuperK Varia) with repetition rates down to 2 MHz. In continuous-wave measurements, the PL was spectrally dispersed by a monochromator (Princeton Instruments Acton SP 2500) and recorded with a nitrogen-cooled silicon CCD (Princeton Instruments PyLoN). Time-resolved PL was detected with avalanche photodiodes (Excelitas SPCM-AQRH or PicoQuant τSPAD).
3
Photoluminescence Decay Measurements of Quantum Dots
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To obtain
the PL decays of the QD or Au/QD solution, a supercontinuum pulsed
laser (Solea, PicoQuant, ∼100–120 ps pulse duration,
2.5 MHz repetition rate) was used to photoexcite each sample at either
510, 530, 550, 570, 580, or 590 nm excitation wavelength. The photoluminescence
of the solutions was acquired through a 20× air objective (Nikon,
N.A. = 0.45) and focused onto a single-photon detector (τ-SPAD,
PicoQuant) that was equipped with the appropriate spectral filter.
The photoluminescence decays were gathered using a time-dependent
single-photon counting module (PicoHarp 300, PicoQuant) with a time
resolution of 32 ps. All experiments were performed under ambient
conditions.
the PL decays of the QD or Au/QD solution, a supercontinuum pulsed
laser (Solea, PicoQuant, ∼100–120 ps pulse duration,
2.5 MHz repetition rate) was used to photoexcite each sample at either
510, 530, 550, 570, 580, or 590 nm excitation wavelength. The photoluminescence
of the solutions was acquired through a 20× air objective (Nikon,
N.A. = 0.45) and focused onto a single-photon detector (τ-SPAD,
PicoQuant) that was equipped with the appropriate spectral filter.
The photoluminescence decays were gathered using a time-dependent
single-photon counting module (PicoHarp 300, PicoQuant) with a time
resolution of 32 ps. All experiments were performed under ambient
conditions.
4
Alternating Laser Excitation Microscopy
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μs-ALEX-experiments were carried out at room temperature (22°C) on a custom-built confocal microscope [18 (link),29 (link)]. In brief, ALEX between 532 and 640 nm was employed with an alternation period of 50 µs, coupled into a confocal microscope, a 60× objective with NA = 1.35 (Olympus, UPLSAPO 60XO) focused the excitation light to a diffraction-limited spot 20 µm into the solution. The excitation intensity amounted to 60 µW at 532 nm (≈30 kW cm−2) and 25 µW at 640 nm (≈25 kW cm−2). Fluorescence emission was collected and spectrally separated onto two APDs (τ-spad, Picoquant, Germany) with appropriate filters (donor channel: HC582/75; acceptor channel: Edge Basic 647LP; AHF Analysentechnik, Germany). The signal was recorded using a custom-written LabView program.
5
Fluorescence Lifetime Imaging of ICG in DMSO
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The fs pulsed laser (PHAROS PH1-10W, LIGHT CONVERSION; repetition rate: 1 MHz; pulse width: 200 fs) beam was introduced into a commercial inverted microscope (IX83, Olympus, Japan) as the excitation light. After being reflected by the dichroic mirror and passing through the objective (PLN20X, Olympus), the laser beam excited the sample. Based on a turnover mirror, emitted fluorescence signals passing through the dichroic mirror and filters were either delivered to a spectrometer (Andor, 193i + iXon DU-897U) or detected by an avalanche photodiode (τ-SPAD, PICOQUANT). The computer with an integrated TCSPC module system (DPC-230 Photon Correlator, Becker & Hickl GmbH) was used to measure the fluorescence lifetime of the sample based on the synchronous signals output by the fs laser and electrical signals from the τ-SPAD. The dependence of the ASF intensity of ICG in DMSO on the intensity of 915 nm fs pulsed laser excitation was derived from ICG’s fluorescence spectra excited at different powers.
6
ISABEL Trap Laser Scanning Protocol
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The ISABEL trap experiments were
performed on a custom-built optical setup as has been recently described31 (link) (see alsoNote S1 and Figure S4 ). Briefly, a near-IR (NIR)
laser at 802 nm is scanned to different x–y positions in the sample plane with a pair of acousto-optic
deflectors (AODs) in a 32-point knight’s tour pattern. The
NIR spot at the sample was ∼500 nm in diameter, and the pitch
of the scan pattern was chosen to provide a time-averaged uniform
intensity of ∼250 kW/cm2 over a 3.6 μm ×
3.6 μm region.31 (link) The backscattered
and reflected light were separated from the pumping beam using a polarizing
beam splitter and quarter-wave plate combination and focused through
an iris to block unwanted light onto a large area photodiode (Newport
2031) for interferometric scattering detection. The fluorescence excitation
laser at 488 nm follows a path distinct from the AODs and is aligned
to overlap with the NIR beam scan pattern using a 775SP dichroic.
This beam illuminates a Gaussian spot at the sample with 1/e2 radius of 5.5 μm, which covers the NIR knight’s tour
pattern. The fluorescence collected in the backward direction is separated
by a 405/488/561/647 quad-pass dichroic, and is passed through a pinhole
equivalent to 3.6 μm diameter in sample space. Green fluorescence
(500–560 nm) is collected on a Si single-photon avalanche photodiode
(τ-SPAD, PicoQuant).
performed on a custom-built optical setup as has been recently described31 (link) (see also
laser at 802 nm is scanned to different x–y positions in the sample plane with a pair of acousto-optic
deflectors (AODs) in a 32-point knight’s tour pattern. The
NIR spot at the sample was ∼500 nm in diameter, and the pitch
of the scan pattern was chosen to provide a time-averaged uniform
intensity of ∼250 kW/cm2 over a 3.6 μm ×
3.6 μm region.31 (link) The backscattered
and reflected light were separated from the pumping beam using a polarizing
beam splitter and quarter-wave plate combination and focused through
an iris to block unwanted light onto a large area photodiode (Newport
2031) for interferometric scattering detection. The fluorescence excitation
laser at 488 nm follows a path distinct from the AODs and is aligned
to overlap with the NIR beam scan pattern using a 775SP dichroic.
This beam illuminates a Gaussian spot at the sample with 1/e2 radius of 5.5 μm, which covers the NIR knight’s tour
pattern. The fluorescence collected in the backward direction is separated
by a 405/488/561/647 quad-pass dichroic, and is passed through a pinhole
equivalent to 3.6 μm diameter in sample space. Green fluorescence
(500–560 nm) is collected on a Si single-photon avalanche photodiode
(τ-SPAD, PicoQuant).
7
Confocal Microscopy with FRET Detection
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Confocal measurements were performed using a MicroTime200 (PicoQuant, Berlin, Germany). The fluorophores were excited using LDH-D-C 485B and LDH-D-C 640B lasers with 485 nm and 640 nm emission (PicoQuant, Berlin, Germany) and a power of 21 μW and 18 μW, respectively. For smFRET and Brightness-gated two-color coincidence detection (BTCCD) measurements, lasers were operated in a pulsed-interleaved excitation (PIE) scheme, in which blue and red excitation was alternated in order to directly excite both channels [22 (link)]. The excitation light was focused and collected by a high numerical aperture water immersion objective (UPLSAPO 60×; Olympus, Hamburg, Germany) and directed through a 75 μm pinhole. The emission signal was separated by a dichroic mirror (T600lpxr, Chroma Technology, Olching, Germany) and filtered by band pass filters of 535 nm (FF01-535/55-25, Semrock, Rochester, NY, USA) and 685 nm (ET685/80m, Chroma Technology, Olching, Germany) for the blue and red channels, respectively. Photons were detected by single-photon avalanche diodes (τ-SPAD, PicoQuant, Berlin, Germany; COUNT-T, Laser Components, Olching, Germany).
8
Spatially-Resolved Photoluminescence Dynamics
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For spatial resolved PL distribution measurement, single crystal sample is excited with CW 532 nm laser (Coherent Verdi) focused with an objective (Nikon CFI Plan Apo Lambda ×60 0.95 NA). PL signal is collected by the same objective and read out with camera (Princeton Instruments, Pixis 1024B). In the confocal PL intensity and lifetime measurements, samples are excited with a 408 nm pulsed laser (MDL 300, PicoQuant) at 40 μm cm−2 pulse energy density (pulse width 180 ps). The PL signal is detected by a single-photon avalanche photodiode (τ-SPAD, PicoQuant), and recorded with a time-correlated single-photon counting system (PicoHarp 300, PicoQuant). As is shown in Supplementary Fig. 5 , the PL intensity went through a fast decay from 0 to 15 ns, and a slow decay after 15 ns. The initial fast drop could be due to two main reasons, bimolecular carrier recombination and carrier diffuse out of the collection area. However, we want to understand the trap-carrier interaction at the TBs, so we focus on the PL intensity decay after 15 ns, which can be well fitted with a single exponential decay model. And no obvious lifetime change can be observed associated with the TBs.
9
Single-molecule multiparameter fluorescence detection
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For single-molecule measurements with multiparameter fluorescence detection, we added 40 µM TROLOX to the measurement buffer to minimize the acceptor blinking and 1 µM unlabeled T4L to prevent any adsorption to the cover glass. A custom-built confocal microscope with a dead time-free detection scheme using 8 detectors (four green (τ-SPAD, PicoQuant, Germany) and four red channels (APD SPCM-AQR-14, Perkin Elmer, Germany)) was used for MFD and fFCS measurements. A time-correlated single photon counting (TCSPC) module with 8 synchronized input channels (HydraHarp 400, PicoQuant, Germany) was used to register the detected photon counts in the Time-Tagged Time-Resolved (TTTR) mode. For more details on TTTR please read69 . The data was analyzed by established MFD procedures31 (link),33 (link),43 (link) and software, a more detailed description is given in Supplementary Methods. Exemplary data analysis is shown in Supplementary Fig. 3 and Supplementary Note 1 , MFD-histograms of all measurements are collected in Supplementary Fig. 2 and Supplementary Fig. 10 .
10
Anti-Stokes Fluorescence Lifetime Measurement
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The anti-Stokes fluorescence lifetime was measured via a time-correlated single-photon counting (TCSPC) system. A 980 nm femtosecond pulsed laser beam was introduced into an inverted microscope. The inside optical path was similar to Section 2.5 . Finally, the anti-Stokes fluorescence signals were extracted and detected by an avalanche photodiode (τ-SPAD, PICOQUANT, Germany). The computer with an integrated TCSPC module (DPC-230 Photon Correlator, Becker & Hickl GmbH, Berlin, Germany) was used to record the fluorescence lifetime of samples based on the synchronous signals output by the femtosecond laser and electrical signals from the τ-SPAD. The fluorescence lifetime is equal to the time when the fluorescence intensity decreases from the peak to one of 1/e (e is the base of the natural logarithm).
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