Lsm upgrade kit

Intensity images as well as fluorescence lifetime imaging microscopy (FLIM) measurements were taken in the confocal mode using an Olympus Fluoview, 1000 (Olympus, Tokyo, Japan) equipped with a time-resolved LSM upgrade kit (PicoQuant GmbH, Berlin, Germany) and a x60 (1.35 N.A.) UPlanSApo oil immersion objective (Olympus). Di-4-ANEPPS was excited at 488 nm using the corresponding line of a multiline argon laser and was detected in the range of 565–665 nm. Images with a frame size of 512 × 512 pixels were acquired. For FLIM measurements, di-4-ANEPPS was excited at 440 nm using a pulsed-laser diode. Fluorescence was detected by a single photon avalanche photodiode (SPAD) and a 620 ± 30 nm bandpass filter was used. Further details of the experimental settings and data analysis can be found in Supplementary Note 3. For every single cell the average lifetime <τ> and the amplitude weighted lifetime $\overline{\mathrm{\tau }}$ were calculated using equations 2 and 3. The values reported in Table 2 are the average of 30–100 cells of at least three independent experiments. The fluorescence lifetime of the probe was independent of the incubation time. The quality of the fitting was judged by the distribution of the residuals and the χ² value.

smFRET pulse interleaved excitation (PIE) experiments were carried out on Zeiss LSM 710 microscope equipped with PicoQuant LSM upgrade kit for time-resolved measurements. As an excitation source two LHD lasers with emission wavelengths of 485 nm and 640 nm were used. The confocal volume was maintained by using water (C-Apochromat 40x/1.2 NA) objective and focusing the emitted light onto a 35 μm pinhole. Donor and acceptor signals were separated from each other by using a second dichroic mirror (545nm) and filters: 520/35 bandpass and 635 longpass, respectively and further recorded by two avalanche photodiodes. For checks of the 30-nt DNA hairpin conformation and PA-Nter processivity, ~40 μl droplet was placed in home-made chambers consisting of cut 0.5 ml Eppendorf tubes glued (Norland Optical Adhesive 63) to a glass cover slip (#1.5, high precision Carl Roth) sealed with parafilm. The measurement was done for an hour in the same buffer as for FCCS, supplemented with 1mM Trolox. The histogram analysis was done using SymPhoTime ver 5.3.2.3 with an implemented PIE_FRET_2D_burst_analysis_05 script (PicoQuant).

FLIM experiments and confocal imaging were carried out using the laser scanning confocal microscope FluoView FV1000 (Olympus Deutschland GmbH, Hamburg, Germany) equipped with a time-correlated single-photon counting (TCSPC) LSM Upgrade Kit (PicoQuant).
For confocal images EGFP and Citrine were excited at 488 nm (argon-ion laser) and mCherry at 561 nm [diode-pumped solid-state (DPSS) laser]. The fluorescence signal was detected by an acousto-optic tunable filter (AOTF) and SIM (simultaneous) scanning unit (Olympus Deutschland GmbH). EGFP and Citrine fluorescence was detected through a dichroic mirror (SDM560) at 505–550 and 515–560 nm respectively. mCherry fluorescence was detected at 590–690 nm.
FLIM images were collected through a 60x/1.35 UPlanSApo oil immersion objective (Olympus Deutschland GmbH). For lifetime microscopy of EGFP or Citrine, the samples were excited with a 470 nm pulsed diode laser (LDH 470, PicoQuant, Berlin, Germany) at a repetition rate of 40 MHz. The photons were collected in a single-photon counting avalanche photodiode (PDM Series, MPD, PicoQuant) and timed using a time-correlated single-photon counting module (PicoHarp 300, PicoQuant) after being spectrally filtered using a narrow-band emission filter (HQ 525/15, Chroma, Bellows Falls). All measurements were carried out in an incubation chamber at 37°C and 5% CO₂.