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Scanless two-photon excitation with temporal focusing


Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics.

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Fig. 1: Schematic representation of 1P and 2P excitation processes.
Fig. 2: Axial propagation of spatially and temporally focused beams.
Fig. 3: Experimental realization of temporal focusing.
Fig. 4: Temporally focused light-shaping methods.
Fig. 5: Temporally focused beams passing through scattering media.
Fig. 6: Temporally focused patterns arranged in 3D.


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We thank R. Sims for proofreading of the manuscript and fruitful discussions on 2PE-based imaging and C. Molinier for the preparation of the supplementary video. We thank the Agence Nationale de la Recherche (grant ANR-15-CE19-0001-01, 3DHoloPAc), the Human Frontiers Science Program (Grant RGP0015/2016), the European Research Council SYNERGY Grant Scheme (HELMHOLTZ, ERC Grant Agreement # 610110), the Fondation Bettencourt Schueller (Prix Coups d’élan pour la recherche française), the Getty Lab, the National Institute of Health (grant NIH 1UF1NS107574-01) and the Axa research funding for financial support.

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Correspondence to Valentina Emiliani.

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Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Fig. 1 Axial propagation of spatially and temporally focused beams.

a, Spatial focusing of a Gaussian high-NA (0.8) and b, low-NA (0.2) beam and corresponding lateral intensity profiles, I, at different axial positions. c, Temporal focusing of a non-spatially focused Gaussian beam and corresponding pulse intensity profile, I, (at z = 0 μm, 1.5 μm and 3 μm), showing the shortening of the pulse duration at the sample plane (NA=0.8).

Supplementary Fig. 2 Comparison of the axial resolution between 2P and 3P temporally focused excited fluorescence.

Reproduced from Toda et al. Measured signal distributions for the 2P-TF (red curve) and 3P-TF (blue curve) excited fluorescence, recorded using a PMT. Fluorescence was excited in a layer of 200-nm fluorescent beads. The exposure times were 100 ms. The input powers for 2P-TF and 3P-TF microscopes were 5.5 mW and 55 mW, and the FWHMs of the signal distributions were 2.1 µm and 1.6 µm, respectively. The out-of-focus excitation for 2P-TF and 3P-TF was estimated as the full width at 1/100 maxima of the signal distributions, which were 69.2 µm and 11.8 µm, respectively, showing a higher out-of-focus signal suppression in 3P-TF by a factor of 5.9. Toda, K. et al. Temporal focusing microscopy using three-photon excitation fluorescence with a 92-fs Yb-fiber chirped pulse amplifier. Biomed. Opt. Express 8, 2796–2806 (2017).

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Supplementary Note 1

Supplemental Video 1

An ultrashort pulse impinges on a dispersive grating at an angle γ (top panel). At each point in time, the intersection between the pulse and the dispersive element is a line, which is scanning the grating surface at a speed of c/sinγ (left, bottom panel) and the sample plane at a de-magnified speed of (c/sinγ)/M (right, bottom panel).

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Papagiakoumou, E., Ronzitti, E. & Emiliani, V. Scanless two-photon excitation with temporal focusing. Nat Methods 17, 571–581 (2020).

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