Stimulated Emission

Mathieu Hautefeuille, Juan A. Hernández-Cordero

Giuseppe Vicidomini, Gaël Moneron, Christian Eggeling +2 more

In stimulated emission depletion (STED) nanoscopy the wavelength of the STED beam is usually tuned towards the red tail of the emission maximum of the fluorophore. Shifting the STED wavelength closer to the emission peak, i.e. towards the blue region, favorably increases the stimulated emission cross-section. However, this blue-shifting also increases the probability to excite fluorophores that have remained in their ground state, compromising the image contrast. Here we present a method to exploit the higher STED efficiency of blue-shifted STED beams while maintaining the contrast in the image. The method is exemplified by imaging immunolabeled features in mammalian cells with an up to 3-fold increased STED efficiency compared to that encountered in standard STED nanoscopy implementations.

Haiyun Qin, Wei Zhao, Chen Zhang +3 more

As one of the most important realizations of stimulated emission depletion (STED) microscopy, the continuous-wave (CW) STED system, constructed by using CW lasers as the excitation and STED beams, has been investigated and developed for nearly a decade. However, a theoretical model of the suppression factors in CW STED has not been well established. In this investigation, the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied. The full-width-at-half-maximum (FWHM) of a CW STED with a doughnut-shaped STED beam is also reanalyzed. It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities. In addition, the FWHM is highly sensitive to both the fluorescence rate (inverse of fluoresce lifetime) and the quenching rate, but insensitive to the rate of vibrational relaxation. For comparison, the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity. Our model is highly consistent with published experimental data for evaluating the spatial resolution. This investigation is important in guiding the development of new CW STED systems.

Galina A. Nemova

Luca Salasnich

Andrew E. S. Barentine, W. E. Moerner

Stimulated emission (StE) remains relatively unused as an image-forming signal despite having potential advantages over fluorescence in speed, coherence, and ultimately resolution. Several ideas for the radiation pattern and directionality of StE remain prevalent, namely, whether a single molecule would radiate StE itself in a pure dipole pattern, or whether its emission direction depends on the driving field. Previous StE imaging has been carried out in transmission, which would collect signal either way. Here, we introduce the StE driving field (the probe) at an angle, using total internal reflection to avoid incident probe light and its specular reflections in our detection path. In this non-collinear detection configuration that also collects some fluorescence from the sample, we observe fluorescence depletion even in the spectral window where an increase in detected signal from StE would be expected if StE radiated like a simple classical dipole. Because simultaneous direct measurement of the fluorescence represents a calibration of the potential size of StE were it spatially patterned like a classical dipole emitter, our study clarifies a critical characteristic of StE for optimal microscope design, optical cooling, and applications using small arrays of emitters.