Materials and Methods

The two-color STED microscope described below is a modified version of the setup described previously (1): The STED light is taken from a regenerative amplified mode-locked Ti:Sapphire oscillator (REGA, Coherent, Palo Alto, CA) providing infrared pulses at 780 nm of ~ 200 fs duration at a 250 kHz repetition rate and with pulse energies of up to 3 μJ. Half of this laser light is used as the far-red STED light, while the other half is fed into an optic parametric amplifier (OPA, 1 Coherent) to generate the visible STED light pulses at 603 nm featuring pulse energies of 100-250 nJ. We stretched the pulse lengths of both STED beams to ~ 300 ps by applying a pair of gratings and a 4m single-mode fiber (Thorlabs, Inc. New Jersey, U.S.A.) for the visible and a 2m hollow-core fiber (Air6-800, Photonic Crystal Fibers, Denmark) for the infrared light. We phase-modified both STED beams to form doughnuts and then overlaid them with the excitation beams delivered by the 470 nm and the 635 nm laser diodes (Picoquant, Berlin, Germany). Each of the pulsed diodes was electronically synchronized with the infrared STED pulse train. PSF-engineering of the STED beams was carried out by spatial light modulators (Hamamatsu, Hamamatsu City, Japan) for each STED beam. Applying a 0-2π helical phase ramp on the wavefronts (2) rendered the doughnut-shaped foci. The four beams were coupled into the microscope by dichroic mirrors (AHF Analysentechnik, Tübingen, Germany); they overfilled the aperture of the 1.4 oil immersion lens used. Fluorescence emission was collected through the same objective and was detected by single photon counting avalanche photodiodes (SPCM-AQR-13-FC, Perkin Elmer Optoelectronics, Canada) with an aperture size corresponding to ~0.8 times the magnified Airy disk of the fluorescence spot