Two-photon light sheet microscopy (2P-SPIM)

Multiphoton fluorescence microscopy has proven invaluable for tissue studies with its ability to provide 3D micron-scale resolution in thick/live samples. However it is a point-scanning technique, which limits its imaging speed to a few images per second (typ. 10^5-10^6 pixels/second). Multiphoton light-sheet imaging is emerging as an efficient method to achieve 50-100x faster two-photon imaging of thick tissues, with the additional benefit of strongly reducing photobleaching.

Left: setup for multicolor two-photon light sheet microscopy using wavelength mixing. Right: 3D reconstruction of cell movements in the beating heart of a zebrafish larva. Image size is 210 * 230 * 300 µm³. Speed scale ranges from 0 to 620 µm/second. Adapted from Mahou et al, 2014.

Related publications

Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency
V. Maioli, A. Boniface, P. Mahou, J. Ferrer Ortas, L. Abdeladim, E. Beaurepaire, W. Supatto
Biomedical Optics Express, 11(10), 6012-6026 (2020).
Fast in vivo imaging of SHG nanoprobes with multiphoton light-sheet microscopy
G. Malkinson, P. Mahou, E. Chaudan, T. Gacoin, A.Y. Sonay, P. Pantazis, E. Beaurepaire, W. Supatto
ACS Photonics, 7(4), 1036-1049 (2020).
Whole-brain functional imaging with two-photon light-sheet microscopy
S. Wolf, W. Supatto, G. Debrégeas, P. Mahou, S.G. Kruglik, J.-M. Sintes, E. Beaurepaire, R. Candelier
Nature Methods 12(5), 379–380 (2015).
Multicolor two-photon light-sheet microscopy
P. Mahou, J. Vermot, E. Beaurepaire, W. Supatto
Nature Methods 11(6), 600-601 (2014).
Advances in whole-embryo imaging: a quantitative transition is underway
P. Pantazis & W. Supatto
Nature Reviews Molecular Cell Biology 15, 327-339 (2014).
Deep and fast live imaging with two-photon scanned light-sheet microscopy
T.V. Truong*, W. Supatto*, D.S. Koos, J.M. Choi, and S.E. Fraser (*equal contributors)
Nature Methods, 8, 757-760 (2011).
Advances in multiphoton microscopy for imaging embryos
W. Supatto, T.V. Truong, D. Debarre, and E. Beaurepaire
Current Opinion in Genetics & Development 21(5) 538-548 (2011).

(Introduction in French)
Microscopie multiphoton illuminée par nappe : imagerie de fluorescence rapide et en profondeur dans les tissus vivants, Supatto, Photoniques (2012).