Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide

Abstract

STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.

Resolution Enhancement and Background Suppression in Optical Super‐Resolution Imaging for Biological Applications

“…Besides the techniques mentioned above based on hardware and light paths, various computational methods were also intensively utilized for improving the STED image quality against background noise, like rolling‐ball background subtraction. [ 32,136–138 ]…”

Challenges facing quantitative large-scale optical super-resolution, and some simple solutions

“…In conventional laser-scanning microscopy, the beam is typically moved using galvanometer-controlled mirrors, whose speed is largely limited by inertia. Several recently implemented alternatives such as fast-oscillating resonant mirrors ( Wu et al., 2015 ), acousto-optic deflectors (AODs) that deflect the beam with sound waves ( Chen et al., 2011 ), or electro-optical deflectors that deflect the beam with electric current ( Schneider et al., 2015 ), have significantly increased the scanning speed. The latter approach was shown to permit a speed of >1,000 frames per second for standard FOV sizes—a rate so high that the pixel dwell times approach the lifetime of the fluorescent states ( Schneider et al., 2015 ).…”

Super‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule

“…Specifically, STED requires a high quality donut achieved through precise optical engineering and alignment (Wu et al 2015). Practically, forming a high quality donut beam necessitates specific microscope design parameters (Lau et al 2012) and careful fine tuning of the alignment of the microscope, usually every time the microscope is used (Saurabh et al 2016)(Fig.…”

“…This requires reducing the laser intensity, typically by adding neutral density (ND) filters.Use these PSFs to fine tune the alignment of the STED microscope (Wu et al 2015). …”