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An evaluation of two‐photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis
Author(s) -
Periasamy Ammasi,
Skoglund Paul,
Noakes Colten,
Keller Raymond
Publication year - 1999
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/(sici)1097-0029(19991101)47:3<172::aid-jemt3>3.0.co;2-a
Subject(s) - two photon excitation microscopy , deconvolution , microscopy , optical sectioning , confocal microscopy , optics , light sheet fluorescence microscopy , confocal , microscope , fluorescence lifetime imaging microscopy , materials science , scanning confocal electron microscopy , fluorescence , physics
The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two‐photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two‐photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two‐photon excitation and confocal microscopy also provide improved rejection of “out‐of‐focus” noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two‐photon images. We have also found that the two‐photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers. Microsc. Res. Tech. 47:172–181, 1999. © 1999 Wiley‐Liss, Inc.