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Role of the Pico-Nano-Second Temporal Dimension in STED Microscopy

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Perspectives on Fluorescence

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 17))

Abstract

In the last decades, several techniques have been developed to push the spatial resolution of far-field fluorescence microscopy beyond the diffraction limit. Stimulated emission depletion (STED) microscopy is a super-resolution technique in which the targeted switching off of the fluorophores by a secondary laser beam results in an effective increase in optical resolution. However, to fully exploit the maximum performances of a STED microscope (effective spatial resolution achievable for a given STED beam’s intensity, versatility, live-cell imaging capability, etc.) several experimental precautions have to be considered. In this respect, the temporal dimension (at the pico- and nanosecond scale) has often a central role on the overall efficiency and versatility of a STED microscope, working in pulsed or continuous-wave mode.

In pulsed STED, temporal alignment between the excitation and STED pulses has direct consequences on the maximum spatial resolution achievable by the STED microscope. In a specific pulsed STED implementation, called single wavelength two-photon excitation STED, the modulation of the temporal width of the pulse results in the use of the very same laser for excitation and depletion of the fluorophores. In continuous-wave (CW)-STED, the analysis of nanosecond fluorescence dynamics allows one to preserve the effective resolution of a STED microscope, but with a significant reduction of the illumination intensity. In this respect, we discuss two different approaches for the analysis of nanosecond dynamics in CW-STED images, namely the so-called gated-STED microscopy and Separation of Photons by LIfetime Tuning (SPLIT)-STED microscopy. Overall, these examples show that concepts developed in time-resolved fluorescence spectroscopy are important for the advancement of optical super-resolution microscopy.

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Authors and Affiliations

  1. Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163, Genoa, Italy

    Luca Lanzanò, Lorenzo Scipioni, Paolo Bianchini & Alberto Diaspro

  2. Department of Computer Science, Bioengineering, Robotics and Systems Engineering, University of Genoa, via Opera Pia 13, 16145, Genoa, Italy

    Lorenzo Scipioni & Marco Castello

  3. Molecular Microscopy and Spectroscopy, Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163, Genoa, Italy

    Marco Castello & Giuseppe Vicidomini

  4. NIC@IIT, Nikon Imaging Center, Istituto Italiano di Tecnologia, via Morego 30, 16163, Genoa, Italy

    Paolo Bianchini & Alberto Diaspro

  5. Department of Physics, University of Genoa, via Dodecaneso 33, 16146, Genoa, Italy

    Alberto Diaspro

Authors
  1. Luca Lanzanò
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  2. Lorenzo Scipioni
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  3. Marco Castello
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  4. Paolo Bianchini
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  5. Giuseppe Vicidomini
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  6. Alberto Diaspro
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Corresponding author

Correspondence to Alberto Diaspro .

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Editors and Affiliations

  1. John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    David M. Jameson

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Lanzanò, L., Scipioni, L., Castello, M., Bianchini, P., Vicidomini, G., Diaspro, A. (2016). Role of the Pico-Nano-Second Temporal Dimension in STED Microscopy. In: Jameson, D. (eds) Perspectives on Fluorescence. Springer Series on Fluorescence, vol 17. Springer, Cham. https://doi.org/10.1007/4243_2016_19

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