Spcm aqr

Confocal fluorescence imaging, FLCS, PCH and FLIM-FRET experiments were performed with a Microtime200 scanning confocal time-resolved microscope system (Picoquant GmbH) (Supplementary Figure S1); A 465 nm picosecond pulsed laser was used to excite the GFP tag and Alexa488 labeled antibodies. Cy3 was excited by a 532 nm laser for in vitro PCH calibration. The excitation beam was delivered to the sample stage through an apochromatic water immersion objective (60×, N.A. = 1.2) and the fluorescence was collected by the same objective, after which the emission was separated by a dual band dichroic (z467/638rpc for blue laser, 49004 DCXR for green laser, Chroma). A 50 μm pinhole was employed to block the off-focus photons and the final signal was additionally filtered by a band-pass filter (520 ± 20 nm for green emission, 610 ± 30 nm for red emission, Chroma) before reaching the single photon avalanche photodiode detectors (SPAD) (SPCM-AQR, PerkinElmer Inc.). Fluorescence information was recorded using the TCSPC (time-correlated single photon counting) module in the time-tagged time-resolved (TTTR) mode (TimeHarp200). Raw fluorescence images and autocorrelation data were first analyzed and then exported with the SymPhoTime software package (PicoQuant GmbH) for further processing. For detailed mathematical fitting and analysis procedure, please refer to the Supplementary Methods.

DNA oligoPAINT FISH imaging was performed at 24 °C in a Leica TCS SP8 confocal microscope equipped with 100× 1.44 NA oil immersion objective (Leica, HC PLAN APO), two SPCM AQR (Perkin Elmer) APD detectors (for Cy5 and Cy3 emission detection) and a HyD Reflected Light Detector (RLD) in gated mode (for Atto 488 emission detection). Oxygen scavenger solution (75mM HEPES-KOH pH7.5, 55mM K-glutamate, 0.9% w/v Glucose, 1mM Ascorbic Acid, 1mM Methyl-viologen, 1×gloxy) was used to protect signals from bleaching(37 (link), 38 (link)). In exposure 1, OligoPAI4NT oligos with Cy5 and Cy3 imaging probes were excited with 551 nm and 649 nm laser light, respectively. The emissions were collected through a dichroic mirror (Shemrock 625 nm edge beamsplitter) in two APD detectors. To avoid further crosstalk between Cy3 and Cy5 emission, we used two band pass filters (Chroma ET595/50m for Cy3, Chroma ET690/50m for Cy5) in front of the two APDs, respectively. In exposure 2, OligoPAINT oligos with Atto 488 imaging probes were excited with 488 nm laser light and the emission was collected in a different path (band pass filter Chroma ET525/50m) on the HyD SMD detector. We used 10%, 1% and 5–7% of maximum laser power to excite Cy3, Cy5 and Atto 488, respectively, selected to avoid bleaching. We scanned an xyz volume ≈5.81×5.81×3 μm³, using 46 nm xy pixel size and 100nm z steps.