Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore

Joanna Oracz,Steffen J Sahl,Volker Westphal,Stefan W Hell,Czesław Radzewicz

doi:10.1038/s41598-017-09902-x

Abstract

In STED (stimulated emission depletion) nanoscopy, the resolution and signal are limited by the fluorophore de-excitation efficiency and photobleaching. Here, we investigated their dependence on the pulse duration and power of the applied STED light for the popular 750 nm wavelength. In experiments with red- and orange-emitting dyes, the pulse duration was varied from the sub-picosecond range up to continuous-wave conditions, with average powers up to 200 mW at 80 MHz repetition rate, i.e. peak powers up to 1 kW and pulse energies up to 2.5 nJ. We demonstrate the dependence of bleaching on pulse duration, which dictates the optimal parameters of how to deliver the photons required for transient fluorophore silencing. Measurements with the dye ATTO647N reveal that the bleaching of excited molecules scales with peak power with a single effective order ~1.4. This motivates peak power reduction while maintaining the number of STED-light photons, in line with the superior resolution commonly achieved for nanosecond STED pulses. Other dyes (ATTO590, STAR580, STAR635P) exhibit two distinctive bleaching regimes for constant pulse energy, one with strong dependence on peak power, one nearly independent. We interpret the results within a photobleaching model that guides quantitative predictions of resolution and bleaching.

Highlights

Stimulated emission depletion (STED) nanoscopy[1] is a well-established method which allows minimally invasive optical access to length scales of tens of nanometers, in fixed and living biological specimens[2]
We investigated the influence of the pulse duration (τ) for short de-excitation pulses (0.13–3 ps), delayed slightly (7 ps) with respect to the ultrashort excitation pulse
For de-excitation pulses comparable in duration to vibrational relaxation of the excited state, the de-excitation efficiency decreases due to unproductive de-excitation/excitation cycles related to the high STED photon flux, before vibrational relaxation can occur[1]

Summary

Introduction

Stimulated emission depletion (STED) nanoscopy[1] is a well-established method which allows minimally invasive optical access to length scales of tens of nanometers, in fixed and living biological specimens[2]. Several chemical approaches have been demonstrated to partially mitigate this problem, including the development of new fluorescent dyes[7, 8], decreasing the concentration of free oxygen to minimize destructive chemical reactions[9], quenching of the triplet state[10], as well as different optical strategies, www.nature.com/scientificreports/ Such as: use of longer STED pulses to avoid multiphoton excitation[11], fast scanners or lasers with low repetition rates to allow triplet state relaxation between two successive pulses[12,13,14], or other approaches which minimize unnecessary exposure to the intense light[15,16,17]. CW de-excitation was not considered in our theoretical modelling This modality, while it features technical simplicity and lower cost of implementation, has the drawback that a constant photon flux acts on the sample at times when all the fluorophores are already in the dark “off ”-state (no fluorescence is expected), which results in increased STED-light-induced fluorescence and bleaching during these times

Methods

Results

Conclusion