Multicolor DNA FISH Imaging Protocol

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.