Iván Coto Hernández

Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation

Stimulation emission depletion (STED) microscopy breaks the spatial resolution limit of conventional light microscopy while retaining its major advantages, such as working under physiological conditions. These properties make STED microscopy a perfect tool for investigating dynamic sub-cellular processes in living organisms. However, up to now, the massive dissemination of STED microscopy has been hindered by the complexity and cost of its implementation. Gated CW-STED (gCW-STED) substantially helps solve this problem without sacrificing spatial resolution. Here, we describe a versatile gCW-STED microscope able to speedily image the specimen, at a resolution below 50 nm, with light intensities comparable to the more complicated all-pulsed STED system. We show this ability on calibration samples as well as on biological samples.

A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy.

Stimulated emission depletion (STED) microscopy is a prominent approach of super-resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time-gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub-diffraction spatial resolution. If the time-gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW-STED microscope. However, time-gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti-Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti-Stokes background. The method hinges on the uncorrelated nature of the anti-Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two-photon-excitation with gated CW-STED microscopy is demonstrated.

Two-Photon Excitation STED Microscopy with Time-Gated Detection

We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture’s complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source.

Influence of laser intensity noise on gated CW-STED microscopy

Stimulated emission depletion (STED) microscopy provides previously unobtainable spatial resolution. But the complexity and cost of the early implementations based on pulsed mode-locked lasers have limited its wide dissemination. The combination of time-gated detection with STED microscopy significantly helped to overcome this limitation. Indeed, a cheap, easy-to-implement and efficient implementation of STED microscopy can be obtained using also continuous-wave (CW) lasers, the so-called gated CW-STED implementation. Here we show that the performance of this system substantially depends on the intensity noise characteristic of the CW laser. Weak intensity fluctuations reduce the average intensity needed for obtaining a certain resolution, which is a key condition to reduce photodamage on the sample. We simulate the influence of intensity noise on the performance of a gated CW-STED microsope and we validate the results comparing two lasers with different intensity noise properties. Finally, we show that the excellent noise characteristics of the optical-pumped-semiconductor lasers allow implementation of a gated CW-STED microscope which provides ~45 nm resolution in a biological sample at moderate (average and peak) intensity light.

Gated‐sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching

The spatial resolution of a stimulated emission depletion (STED) microscope is theoretically unlimited and practically determined by the signal‐to‐noise ratio. Typically, an increase of the STED beams power leads to an improvement of the effective resolution. However, this improvement may vanish because an increased STED beams power is often accompanied by an increased photobleaching, which worsen the effective resolution by reducing the signal strength. A way to lower the photobleaching in pulsed STED (P‐STED) implementations is to reduce the peak intensity lengthening the pulses duration (for a given average STED beams power). This also leads to a reduction of the fluorophores quenching, thus a reduction of the effective resolution, but the time‐gated detection was proved to be successful in recovering these reductions. Here we demonstrated that a subnanosecond fiber laser beam (pulse width ∼600 ps) reduces the photobleaching with respect to a traditional stretched hundreds picosecond (∼200 ps) beam provided by a Ti:Sapphire laser, without any effective spatial resolution lost.

Gated STED microscopy with time-gated single-photon avalanche diode

Stimulated emission depletion (STED) microscopy provides fluorescence imaging with sub-diffraction resolution. Experimentally demonstrated at the end of the 90s, STED microscopy has gained substantial momentum and impact only in the last few years. Indeed, advances in many fields improved its compatibility with everyday biological research. Among them, a fundamental step was represented by the introduction in a STED architecture of the time-gated detection, which greatly reduced the complexity of the implementation and the illumination intensity needed. However, the benefits of the time-gated detection came along with a reduction of the fluorescence signal forming the STED microscopy images. The maximization of the useful (within the time gate) photon flux is then an important aspect to obtain super-resolved images. Here we show that by using a fast-gated single-photon avalanche diode (SPAD), i.e. a detector able to rapidly (hundreds picoseconds) switch-on and -off can improve significantly the signal-to-noise ratio (SNR) of the gated STED image. In addition to an enhancement of the image SNR, the use of the fast-gated SPAD reduces also the system complexity. We demonstrate these abilities both on calibration and biological sample. The experiments were carried on a gated STED microscope based on a STED beam operating in continuous-wave (CW), although the fast-gated SPAD is fully compatible with gated STED implementations based on pulsed STED beams.

The importance of the photon arrival times in STED microscopy

In a stimulated emission depletion (STED) microscope the region from which a fluorophore can spontaneously emit shrinks with the continued STED beam action after the excitation event. This fact has been recently used to implement a versatile, simple and cheap STED microscope that uses a pulsed excitation beam, a STED beam running in continuous-wave (CW) and a time-gated detection: By collecting only the delayed (with respect to the excitation events) fluorescence, the STED beam intensity needed for obtaining a certain spatial resolution strongly reduces, which is fundamental to increase live cell imaging compatibility. This new STED microscopy implementation, namely gated CW-STED, is in essence limited (only) by the reduction of the signal associated with the time-gated detection. Here we show the recent advances in gated CW-STED microscopy and related methods. We show that the time-gated detection can be substituted by more efficient computational methods when the arrival-times of all fluorescence photons are provided.

Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)

Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.

STED Microscopy with Time-Gated Detection:Benefits and Limitations

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used [1] to substantially improve the effective spatial resolution in STED nanoscopy using pulsed excitation, continuous wave (CW) STED beams and by sorting photons depending by them arrival-times (time-gated detection). We present theoretical/experimental data that characterize the time evolution of the effective detection volume of a STED microscope and illustrate the physical basis, the benefits, and the limitations of this new STED implementation, namely gated CW-STED (gCW-STED). Among all the STED implementations, gCW-STED provides the highest effective resolution at low light intensity and is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also strongly reduces the influence of local variations of the fluorescence lifetime on STED microscopy resolution. Finally, combination of gCW-STED with fluorescent correlation spectroscopy (FCS) is discussed: gCW-STED offers the unique property of tuning the effective detection volume by sorting photons in time.[1] Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, et al. (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8: 571-573.