Accurate Background Reduction in Adaptive Optical Three-Dimensional Stimulated Emission Depletion Nanoscopy by Dynamic Phase Switching

Abstract

Stimulated emission depletion (STED) fluorescence nanoscopy allows the three-dimensional (3D) visualization of nanoscale subcellular structures, providing unique insights into their spatial organization. However, 3D-STED imaging and quantification of dense features are obstructed by the low signal-to-background ratio (SBR) resulting from optical aberrations and out-of-focus background. Here, combining adaptive optics elements, we present an easy-to-implement, flexible, and effective method to improve the SBR by dynamic phase switching. By switching to a counterclockwise vortex phase mask and a top-hat one with an incorrect inner radius, the depletion pattern features a nonzero-intensity center, enabling accurate background recordings. When the recorded background is subtracted from the aberration-corrected 3D-STED image, the SBR in dense sample areas can be improved by a factor of 3−7 times. We demonstrate our method on various dense subcellular structures, showing more advantages than the software-based background subtraction algorithms.

Three-dimensional multi-color optical nanoscopy at sub-10-nm resolution based on small-molecule organic probes

“… 2 To resolve this dilemma when imaging thicker, we employed a 1.35-NA silicon-oil objective and further integrated adaptive optics configuration into our instrument to dynamically compensate for the sample-induced aberrations. 12 This scheme ensures that sub-10-nm resolution remains almost constant throughout the whole imaging process.…”

“…The STED images were obtained using a custom 3D-STED nanoscope 12 equipped with an Apochromat 100×/1. 35 numerical aperture, silicone-oil objective lens (Olympus).…”

“…To allow nearly isotropic resolution, the STED xy beam power is 1/4 that of the STED z beam. 12 A double-pass spatial light modulator (SLM, X13139-02, Hamamatsu) configuration 27 is used to encode the vortex or top-hat phase on each polarization component.…”

Photon-Weight-Reallocated Stimulated Emission Depletion Nanoscopy

“…The subtraction coefficient γ is related to the occupancy of the focus and defocus components around the image. In DPS-STED, the least-squares method was employed to determine the optimal γ value, that is, by minimizing the value of normal|| normalS 0 − γ × B 1 normal|| 2 2 . The assessment method should be also suitable to get the optimal subtraction coefficient γ of HDS.…”

“…16 However, these methods cannot compensate for background disturbances, making them poor at background suppression. The second category comprises techniques for optimizing optical methods and incorporating hardware systems to directly collect and remove background noise including double-depletion STED, 17 polarization switching STED, 18 dual-phase STED (DPS-STED), 19 and isotropic STED based on a multidetector 4pi system. 20 Nonetheless, these methods require optically complex and cumbersome imaging processes, making them unfriendly to users and samples.…”