Increased penetration depth by non-degenerate two-photon microscopy

Mu-Han Yang; Sanaz Sadegh; Martin Thunemann; Payam Saisan; Anna Devor; Yeshaiahu Fainman

doi:10.1117/12.2566999

20 August 2020 Increased penetration depth by non-degenerate two-photon microscopy

Mu-Han Yang, Sanaz Sadegh, Martin Thunemann, Payam Saisan, Anna Devor, Yeshaiahu Fainman

Proceedings Volume 11497, Ultrafast Nonlinear Imaging and Spectroscopy VIII; 114970X (2020) https://doi.org/10.1117/12.2566999
Event: SPIE Optical Engineering + Applications, 2020, Online Only

Abstract

In classical Two photon microscopy (TPM), fluorescence excitation happens via absorption of two photons with the same energy. However, the energies of the two photons do not need to be the same: the sum of their energies must be equal to the total energy required for the ground state to excited state transition. This feature allows for non-degenerate two-photon excitation (ND-TPE), where excitation occurs via simultaneous absorption of two photons of different energies derived from two laser beams. ND-TPE has been exploited in fluorescence microscopy to extend the range of excitation wavelengths , increase resolution, increase penetration depth, and mitigate excitation outside of the focal volume.We use non-degenerate two-photon excitation where the two excitation beams are displaced in space outside the focal volume to increase the signal-to-background ratio (SBR), overcoming the fundamental penetration depth limit of conventional two-photon microscopy.

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Citation Download Citation

Mu-Han Yang, Sanaz Sadegh, Martin Thunemann, Payam Saisan, Anna Devor, and Yeshaiahu Fainman "Increased penetration depth by non-degenerate two-photon microscopy", Proc. SPIE 11497, Ultrafast Nonlinear Imaging and Spectroscopy VIII, 114970X (20 August 2020); https://doi.org/10.1117/12.2566999

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KEYWORDS

Two photon excitation microscopy

Photons

Absorption

Brain

Point spread functions

Tissues

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