Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples

Abstract

Stimulated emission depletion microscopy (STED) is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of specimens’ optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the sever distortion of the depletion beam profile may cause complete loss of the super resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is hard to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique (COAT). The full correction can effectively maintain and improve the spatial resolution in imaging thick samples.

Low‐Saturation‐Intensity, High‐Photostability, and High‐Resolution STED Nanoscopy Assisted by CsPbBr3 Quantum Dots

“…The emission fluorescence wavelengths between 510 and 570 nm were collected on a time‐gated hybrid detector. For details of the light path map, the reader is referred to previous work by the authors …”

“…In particular, researchers in super‐resolution imaging won the 2014 Nobel Prize in Chemistry for “the development of super‐resolved fluorescence microscopy”. STED is one of the major super‐resolution imaging techniques in fast super‐resolution imaging and stands out from other such methods, its unique and straightforward approach (pure optical methods) can achieve super‐resolution images without postreconstruction and chemical reactions . In common STED nanoscopy implementations, two laser beams are needed.…”

Biophotonics in China

“…Recently, they used phasor‐FLIM to directly measure fluorescence lifetimes from H&E‐stained sections and proposed for the first time to develop a further utilization of eosin for fluorescence lifetime encoded multitarget imaging . They also proposed to use the coherent optical adaptive technique (COAT) to correct the distorted wavefront of a Stimulated Emission Depletion STED beam in a STED microscope so that the imaging performance of STED for thick biological samples has been improved .…”

Numerically enhanced adaptive optics-based 3D STED microscopy for deep-tissue super-resolved imaging

“…In single molecule localization microscopy, utilization of self-interference allowed for imaging at 50 µm depth with 100nm localisation precision 33 , while application of adaptive optics enabled imaging through 100 µm of tissue in the central nervous system of a Drosophila with a lateral localization precision of 143nm 34 . Lateral STED imaging through thick samples has been successfully demonstrated using Laguerre-Gaussian (LG) depletion beams [35][36][37] as well as Bessel depletion beams for imaging fluorescent beads at depths as much as 155 µm 38 . Adaptive optics approaches have been implemented to image a rat heart at a depth of 36µm 37 while a glycerol immersion objective lens with correction collar was used to facilitate imaging brain synapses at 120µm 39 .…”