Super-Resolution Imaging of Nuclear Bodies by STED Microscopy

AbstractWe demonstrate bleaching-independent STED microscopy using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging in fixed and living cells. Foremost, we achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy with up to 100 min acquisition time.

Download Full-text

Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging

Biosensors ◽

10.3390/bios11090330 ◽

2021 ◽

Vol 11 (9) ◽

pp. 330

Author(s):

Jia Zhang ◽

Xinwei Gao ◽

Luwei Wang ◽

Yong Guo ◽

Yinru Zhu ◽

...

Keyword(s):

Low Power ◽

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Live Cell ◽

Stimulated Emission ◽

Live Cells ◽

Sted Microscopy ◽

Resolution Imaging ◽

Stimulated Emission Depletion

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

Download Full-text

Aberration correction for improving the image quality in STED microscopy using the genetic algorithm

Nanophotonics ◽

10.1515/nanoph-2018-0133 ◽

2018 ◽

Vol 7 (12) ◽

pp. 1971-1980 ◽

Cited By ~ 5

Author(s):

Luwei Wang ◽

Wei Yan ◽

Runze Li ◽

Xiaoyu Weng ◽

Jia Zhang ◽

...

Keyword(s):

Genetic Algorithm ◽

Wave Front ◽

Super Resolution ◽

Stimulated Emission ◽

Aberration Correction ◽

Sted Microscopy ◽

Laser Wave ◽

Resolution Imaging ◽

Compensation Approach

AbstractWith a purely optical modulation of fluorescent behaviors, stimulated emission depletion (STED) microscopy allows for far-field imaging with a diffraction-unlimited resolution in theory. The performance of STED microscopy is affected by many factors, of which aberrations induced by the optical system and biological samples can distort the wave front of the depletion beam at the focal plane to greatly deteriorate the spatial resolution and the image contrast. Therefore, aberration correction is imperative for STED imaging, especially for imaging thick specimens. Here, we present a wave front compensation approach based on the genetic algorithm (GA) to restore the distorted laser wave front for improving the quality of STED images. After performing aberration correction on two types of zebrafish samples, the signal intensity and the imaging resolution of STED images were both improved, where the thicknesses were 24 μm and 100 μm in the zebrafish retina sample and the zebrafish embryo sample, respectively. The results showed that the GA-based wave front compensation approach has the capability of correction for both system-induced and sample-induced aberrations. The elimination of aberrations can prompt STED imaging in deep tissues; therefore, STED microscopy can be expected to play an increasingly important role in super-resolution imaging related to the scientific research in biological fields.

Download Full-text