Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach

Abstract

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications. In principle, STED super resolution is obtained by stimulated emission partially inhibiting the spontaneous emission in the periphery of a diffraction-limited area. However, very high depletion laser power is generally necessary for the enhancement of imaging resolution, which is harmful to live biological specimens due to its high phototoxicity and photo-bleaching effects. Therefore, further improving the STED resolution at a lower depletion power level has recently attracted increasing interest from researchers in various fields. In this work, a phasor plot approach combined with fluorescence lifetime imaging microscopy (FLIM) is used to resolve the abovementioned problem based on a long- and short-lifetime criterion. Firstly, the time-resolved data obtained by STED-FLIM is converted to the frequency domain via a phasor approach. Next, partial data is extracted according to the information on the phase and amplitude for resolution improvement. Then, fluorescent microspheres (100 nm in diameter) are observed under different depletion powers, resulting in a series of improved resolution through phasor plots. Finally, this method is applied to image human Nup153 in fixed HeLa cells, providing a 86 nm higher resolution than that in traditional STED imaging at a depletion power of 20 mW.

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam

“…Increased power exacerbates photo-damage, a concern in STED microscopy which led to technical advances [47–51] and conceptual generalizations for the use of an optical doughnut [52,53]. Still, whenever a doughnut is used, the highest intensities at the doughnut crest, which are evidently the most photo-damaging for the sample, are too far away from the center to contribute significantly to resolution improvement [50].…”

Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy

“…The fluorescence lifetime of most photons in the excited state has changed in STED imaging mode due to the stimulated emission effect, which means it needs multiexponential curve to fit the fluorescence lifetime. However, the fluorescence lifetime of STED images can be treated as a combination of long lifetime and short lifetime, which represented the spontaneous emission effect and the stimulated emission effect from the central region and the annular region of the depletion beam, respectively [21]. Therefore, double exponential curve can not only fit the lifetime very well but also simplify the processing.…”

Photon-separation to enhance the spatial resolution of pulsed STED microscopy

“…Notably, the method proposed in ref. 18 better selects the two regions in the phasor plot ("abandoned" and "selected" areas), since it allows considering all the pixels contained in the semicircle, but the final pSTED result is still a segmented version of the raw image. Importantly, the nonlinear nature of the segmentation operation (i.e.…”

“…A first method based on the phasor analysis of a pSTED image has been developed by us 17 and successively improved by Wang L. et al 18 This method uses the phasor-plot representation of the pSTED image to generate a pixel segmented version of the raw data; the segmentation selects the pixels characterised by a slow fluorescence temporal dynamic, thus composed primarily of "wanted" photons, and discards the pixels identified by a fast fluorescence temporal dynamic, thus composed primarily of "unwanted" photons. In contrast, the SPLIT approach selects from each pixel only the "wanted" photons, rather than performing a simple binary pixel classification.…”