MINSTED nanoscopy enters the Ångström localization range

nature biotechnology Article https://doi.org/10.1038/s41587-022-01519-4 MINSTED nanoscopy enters the Ångström localization range Received: 22 March 2022 Accepted: 20 September 2022 Michael Weber 1,6, Henrik von der Emde 1,6, Marcel Leutenegger1, Philip Gunkel 2, Sivakumar Sambandan1,3,4, Taukeer A. Khan1, Jan Keller-Findeisen1, Volker C. Cordes 2 and Stefan W. Hell 1,5 Published online: xx xx xxxx Check for updates Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power. By blue-shifting the STED beam and separating fluorophores by on/off switching, individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Because our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up new areas of application in the study of macromolecular complexes in cells. Since the 1970s, fluorescence microscopy has been indispensable for studying the distribution of biomolecules in cells. At the turn of this century, STED microscopy1 broke the diffraction barrier that imposed an apparently unsurmountable physical limit on optical resolution, opening up the imaging of cells at the tens of nanometers scale. This transformation has become possible by relying on the on/off switching of the ability of fluorophores to fluoresce. The recently introduced MINFLUX2 and MINSTED3 nanoscopy added another factor of ten, thus finally reaching a resolution at the scale of the fluorescence labels. MINFLUX and MINSTED uniquely combine the specific strongpoints of STED and the method called PALM/STORM4. Like the latter, they switch the fluorescence ability individually per fluorophore, ensuring the finest possible discrimination of neighboring fluorophores. However, unlike in PALM/STORM, where the stochastic, initially unknown position of the fluorophore is derived from the diffraction spot of fluorescence detections emerging on a camera, in MINFLUX and MINSTED the individual fluorophores are localized with a movable reference point in the sample that is usually defined by the intensity minimum of a donut-shaped beam. By moving this donut minimum closer to the position of the fluorophore during the localization process, MINFLUX and MINSTED increase the information gain per detected photon so that precisions of σ ≈ 1–2 nm are routinely attained with only 200–1,000 photons on single fluorophores. Clearly, once 3σ < 1 nm and the molecular construct linking the fluorophores to the target biomolecules is controlled, structural biology type of studies inside cells should become viable using optical microscopes. A major factor limiting the attainable precision is background— that is, photon detections not stemming from the target fluorophore. Initial experiments have shown that MINSTED has an advantage over MINFLUX in this regard, because its donut-shaped STED beam is 1 Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany. 2Department of Cellular Logistics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany. 3Synaptic Metal Ion Dynamics and Signaling, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany. 4Laboratory of Neurobiology, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany. 5 Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany. 6These authors contributed equally: Michael Weber, Henrik von der Emde. e-mail: Nature Biotechnology Article https://doi.org/10.1038/s41587-022-01519-4 a b 100 Excitation Direct excitation Effectiv e PSF STED + 50 Absorption 500 Emission 550 600 650 0 700 Wavelength λ (nm) Cy3B 636 nm 28 % 5% Atto 647N 775 nm c STED wav elength d Pump ON OFF S1 S0 1.4 ns DM1 Pr3+ doped fiber amplifier 450 nm Phase plate DM3 MINSTED localization PH 636 nm λ/4 Sample space EOD scanner DET Standard STED imaging STED DM2 EXC 560 nm 200 ps Objective 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 quarterwave plate (λ/4) ensures circular polarization. Fluorescence collected from the sample is de-scanned, spatially filtered by a pinhole (PH) and detected. designed to suppress fluorescence. This is contrary to MINFLUX where the donut elicits fluorescence in an area that is about three times larger than in a standard confocal microscope. Besides, building on a STED microscope that inherently offers resolution tuning by changing the donut power, a MINSTED setup can readily accommodate a resolution ranging from the diffraction limit to the molecular scale. Nonetheless, our initial MINSTED study revealed that subtle heating by the STED beam, probably of the sample and the lens immersion oil, limits the precision to σ > 1 nm. This is because the popular STED beam of wavelength λSTED = 775 nm entails a several orders of magnitude higher average power than what is typically used in confocal and MINFLUX microscop (...truncated)