Open AccessTwo-photon PSF-engineered image scanning microscopy
Author(s) -
Omer Tzang,
Dan Feldkhun,
Anurag Agrawal,
Alexander Jesacher,
Rafael Piestun
Publication year - 2019
Publication title -
optics letters/optics index
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.524
H-Index - 272
eISSN - 1071-2763
pISSN - 0146-9592
DOI - 10.1364/ol.44.000895
Subject(s) - optics , point spread function , femtosecond , materials science , microscopy , biological imaging , holography , two photon excitation microscopy , penetration depth , iterative reconstruction , physics , fluorescence , laser , computer science , artificial intelligence
We present two-photon fluorescence image scanning microscopy (ISM) with engineered excitation and detection point-spread-functions enabling 3D imaging in a single 2D scan. This demonstration combines excitation using a holographic multispot array of focused femtosecond pulses with a high-efficiency single-helix PSF phase mask detection. Camera detection along with a multiview reconstruction algorithm allows volumetric imaging of biological samples over a depth of field spanning more than 1500 nm with an axial resolution of better than 400 nm. The nonlinear two-photon process improves sectioning and the inherent longer wavelengths increase the penetration depth in scattering samples. Our method extends the performance of 3D ISM towards thicker biological samples.