Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Abstract

Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.fluorescence nanoscopy | STED microscopy | photobleaching | superresolution

Adaptive-illumination STED nanoscopy

“…The 2D and 3D DyMIN STED fluorescence nanoscopy was demonstrated with a microscope setup extended from the one previously described in ref. 11. For sample preparation of rat hippocampal neurons (Fig.…”

“…It enables the recording of many more frames of the respective sample region and/or imaging at higher resolutions, since a higher STED intensity can be applied, albeit at the expense that neighboring areas are potentially negatively affected (11).…”

“…But the fluorophores, as they are located near the minimum, do not experience the high intensity of the doughnut crest. This is exactly the rationale behind MINFIELD (11), but here the dynamic probing and adjustment of the STED intensity lifts the limitation of small scan fields, while it still avoids scanning a fullpower doughnut over the whole sample.…”

“…In RESCue, judgment of a region to be empty is followed by immediate shutdown of all beams at scan positions that are not occupied by fluorophores. Finally, a method called MINFIELD (11) was recently presented, which works by limiting the scan field to a region of about the same size as the doughnut minimum "valley." This avoids scanning the high-intensity crests over the fluorophores, significantly reducing their bleaching.…”

Breaking the Axial Diffraction Limit: A Guide to Axial Super‐Resolution Fluorescence Microscopy

“…To simplify the system, only one SLM was adopted to modulate the same beam twice, so as to produce 3D depletion on the basis of the particular polarization‐response characteristic of SLM . To further improve the 3D resolution of STED, which is mainly limited by photobleaching and photodamage, the focal intensity distributions were adaptively controlled by the SLM to reduce the overall illumination dose and achieve ≈60‐nm axial resolution . Axial depletion can also be used for other similar point scanning techniques, such as fluorescence emission difference microscopy, to increase their resolution …”