2PE-STED Microscopy with a Single Ti: Sapphire Laser for Reduced Illumination

Qifeng Li,Yang Wang,Sherry S H Wu,Da Chen,Irene Georgakoudi

doi:10.1371/journal.pone.0088464

Abstract

We reported a new effective approach to carry out two-photon excitation stimulated emission depletion (2PE-STED) microscopy using a single Ti:sapphire laser system. With an acoustic-optic Bragg cell, the modulated-CW 2PE STED microscope had the benefits of both CW and pulse approaches: lower input power, simple optical scheme and no complicated synchronization. Additionally, it also took advantages of fluorescence yield increasing. The sub-diffraction-limit resolution was demonstrated using ATTO 425-tagged clathrin-coated vesicles.

Highlights

Fluorescence microscopy is one of the most powerful techniques available for biological studies. [1] because of the diffraction limit, the resolution of a far-field fluorescent microscope is typically no better than 200 nm
Earlier stimulated emission depletion (STED) microscopes used two synchronized trains of pulses: one excitation pulse of typically less than 100 ps duration followed by a 200 ps pulse for depletion. [10,11,12] In these setups, the depletion pulses in the visible region were typically generated in an optical parametric oscillator (OPO), stretched to 200 ps, and synchronized with the excitation pulse
It has been demonstrated that reducing the repetition rate below 1 MHz increased the total 2PE fluorescence yield by,25-fold for GFP and,20-fold for ATTO 532

Summary

Introduction

Fluorescence microscopy is one of the most powerful techniques available for biological studies. [1] because of the diffraction limit, the resolution of a far-field fluorescent microscope is typically no better than 200 nm. [1] because of the diffraction limit, the resolution of a far-field fluorescent microscope is typically no better than 200 nm. EM has very high resolution, [2] it has many practical issues that limit its utility for biological studies. Recent developments in super resolution optical microscopy, such as stimulated emission depletion (STED) [4], photoactivated localization microscopy (PALM) [5,6], stochastic optical reconstruction microscopy (STORM) [7], and structure illumination microscopy (SIM) [8] have achieved sub-diffraction-limit resolution. [9] While SIM, PALM, and STORM require mathematical reconstructions to obtain a high-resolution image, STED does not. Earlier STED microscopes used two synchronized trains of pulses: one excitation pulse of typically less than 100 ps duration followed by a 200 ps pulse for depletion. It was shown that STED microscopy can be implemented with CW lasers, simplifying the instrumental requirement for STED microscopy. [13]

Methods

Results

Conclusion