Fig. 1: Blue-shifted MINSTED.

a, Qualitative fluorescence and absorption spectra of the fluorophore Cy3B, including our selection of wavelength for excitation (560 nm, green) and de-excitation by stimulated emission (636 nm, red). Reaching well into the emission peak, the cross-section for stimulated emission amounts to 28% of its global maximum, at the expense of slight ‘direct’ excitation of ground state Cy3B fluorophores by the donut-shaped STED beam (inset). b, Blue-shifting the wavelength of the donut (lower donut has shorter wavelength) for a given power sharpens the central peak of the effective PSF of the STED microscope but gives rise to a pedestal. c, The pedestal leads to weak fluorescence from bystander fluorophores, thus compromising the contrast in standard STED imaging (left). Because only one fluorophore is active in MINSTED, the pedestal is ineffectual (right), meaning that the benefits of the blue-shifted STED wavelength can be exploited. d, Schematic of the MINSTED setup: originating from a 636-nm emitting laser diode, the STED 1.4-ns pulses are amplified by a Pr³⁺ doped fiber pumped with 450-nm laser diode, deflected by a dichroic mirror (DM1), converted into a donut by a phase plate and aligned with a laser emitting 200-ps pulses for excitation at 560 nm. The co-aligned beams are steered in the focal plane of the objective lens by an EOD, whereas the quarter-wave plate (λ/4) ensures circular polarization. Fluorescence collected from the sample is de-scanned, spatially filtered by a pinhole (PH) and detected.