Stimulated emission depletion microscopy has been used to overcome the diffraction limit of confocal fluorescence microscopy. By exploiting information present in the arrival time of fluorescence photons through time-gating, the resolution of STED microscopes can be improved significantly. The resolution improvement of this technique - termed "T-STED" - becomes most evident in CW-STED where the STED beam is of long duration compared to the lifetime of the fluorophore.

Introduction

Until recent years, the diffraction limit has dictated the resolution performance of fluorescence microscopy [1, 2]. Several new techniques have recently emerged to bypass this fundamental far-field limit [1-3]. One of these techniques is called stimulated-emission depletion (STED) microscopy [4]. In STED microscopy, excited fluorophores are forced to emit via stimulated emission by a depletion beam, termed the STED beam. To form an image an excitation beam and the superimposed STED beam are raster-scanned across the sample and the fluorescence is detected much like in a regular confocal laser-scanning microscope. However, the intensity distribution of the STED beam is shaped such that only molecules in the periphery of the focus are affected by the STED beam and only molecules towards the centre emit via spontaneous emission. Increasing the STED beam intensity increases the probability of fluorophores to emit via stimulated emission thus squeezing the area in which fluorophores are left to emit via spontaneous emission from the periphery towards the centre of the PSF. Thus, the effective extent of the point-spread function (PSF) can be reduced dramatically. The result is a sharper PSF and resolution below the diffraction limit. This process is the basis for any STED microscope - pulsed or continuous wave (CW) - in use today [5].

However, when a second decay channel for relaxation to the ground state is present, the rate of de-excitation and thus the lifetime of the fluorophore is changed as well [6] - a fact that has been ignored in current STED microscopes. In a recent publication we calculated the change of fluorescence lifetime for varying STED beam intensities [7], and we showed that photon arrival times contain spatial information.

More importantly, this information can be extracted by time-gating the detected signal. In doing so, timed-gated STED (T-STED) microscopy increases the resolution of a standard STED microscope without requiring additional laser power. In an independent publication Vicidomini et al. recently provided the first experimental verification of this resolution improvement [8].

In the following we provide a short overview of how T-STED works and discuss the performance increase that can be expected in different STED microscope setups based on our detailed analytical calculations to be found in reference 7.

Spatial Information Encoded in Photon Arrival Times

In order to explain the nature of T-STED we start by assuming a set of excited fluorophores which is illuminated by a STED beam with a Laguerre-Gaussian intensity distribution, LG01 („donut mode"; see figure 1a). The probability of excited fluorophores to emit via fluorescence in presence of the STED beam is dependent on its local intensity (figure 1a). However, the lifetime of the excited fluorophores at various positions also changes dependent on the local depletion beam intensity (figure 1b). At the STED beam centre its intensity is zero and hence the fluorophore lifetime remains unchanged (blue circle figure 1a; blue graph figure 1b). At positions which coincide with the intensity maximum of the STED beam the fluorophore lifetime is reduced (orange circle figure 1a; orange graph figure 1b). Thus, the earliest photons to arrive are most likely from fluorophores located in regions of highest STED beam intensity, and the last photons to arrive are most likely from fluorophores located in the central minimum (figure 1b).
By recording the arrival time of photons when imaging it is thus possible to increase image resolution by constructing the final image only from photons which arrive after a certain time; i.e. early photons from the periphery of the effective PSF are selectively discarded.

T-STED in a CW-STED Microscope Configuration

STED systems can be operated with depletion beams that are either pulsed or CW; however, time-gating provides the most benefit for CW STED beams. Here we assume a pulsed excitation laser beam with a repetition period, tE, much larger than the unstimulated fluorophore lifetime τ (fig. 2a). The resolution can be improved arbitrarily in theory by increasing the time-gate period, tG, which equals to raising the STED beam pulse energy simply by integrating it over a longer period (fig. 2c, d). Thus a higher „effective" STED beam energy will result in better resolution. However, one cannot wait indefinitely. Fluorophores from the central STED minimum also decay; thus, time-gates much larger than the unstimulated fluorophore lifetime throw away many of the photons actually needed for the image. Hence, the cost of the resolution enhancement with time-gating is image brightness. The use of fluorophores with long lifetimes will alleviate this signal reduction, allowing the longest possible time-gate for best resolution. The advantages of CW-T-STED are very obvious. Using relatively low power and cheap sources the user can build a high performance STED microscope. Recently, the power of the CW-T-STED scheme was demonstrated by Vicidomini et al. who could reduce the depletion power by more than 50% and still yield better resolution than in high-power experiments [8; termed G-STED by Vicidomini et al.].

Moreover, in a CW-T-STED arrangement, resolution can be adjusted offline after image acquisition simply by constructing the final image from photons which arrived at the detector after a user-definable time-gate. The user then has the choice to find the optimal setting of the time-gate, which is a trade-off between better resolution and dimmer images.

T-STED in Pulsed STED Microscopes

When using a pulsed laser for the depletion beam, the upper limit of resolution is ultimately set by the pulse energy of the source and is valid only for an infinitesimally short pulse duration since during the STED pulse, fluorescence emission already takes place (figure 2b). Stretching the pulse width will reduce the resolution at a given pulse energy. In our recent publication we showed that the loss in resolution due to finite pulse lengths can be restored by using a time-gate period, tG, equal in length to the STED beam pulse duration, tS (tG = tS) [7]. Analogously to the CW-T-STED configuration image brightness is reduced by applying the time-gate.

Apart from improving the resolution of current STED microscopes, pulsed T-STED also allows the user to stretch the depletion pulse width to avoid phototoxic effects and photobleaching introduced by the depletion laser while at the same time maintaining the resolution of the microscope. This might be beneficial for live cell imaging applications.

In summary our findings show that the performance of current STED microscopes can be significantly improved by taking into account the spatial information present in the arrival times of spontaneous emission. In both STED con­fi­gurations, CW or pulsed, time-gating the detection exploits this information, resulting in an enhancement of resolution [7].

Acknowledgements
Funding was provided by Nanosystems Initiative Munich and the European Research Council.

References
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[3] Rust M.J. et al.: Nature Methods 3 (10) 793-796 (2006)
[4] Hell S.W. et al.: Optics Letters 19 (11) 780-782 (1994)
[5] Willig K.I. et al.: Nature Methods 4 (11) 915-918 (2007)
[6] Lakowicz J.R. et al.: Journal of Biomedical Optics 13 (2) 029901 (2008)
[7] Moffitt J. et al.: Optics Express 19 (5) 4242-4254 (2011)
[8] Vicidomini G. et al.: Nature Methods 8, 571-573 (2011)