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Imaging of optically thick specimen using two‐photon excitation microscopy
Author(s) -
Gerritsen H.C.,
De Grauw C.J.
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<206::aid-jemt6>3.0.co;2-h
Subject(s) - two photon excitation microscopy , oil immersion , microscopy , excitation , materials science , fluorescence , optics , immersion (mathematics) , microscope , confocal , fluorescence microscope , fluorescence lifetime imaging microscopy , confocal microscopy , optical microscope , numerical aperture , excitation wavelength , chemistry , wavelength , scanning electron microscope , optoelectronics , physics , composite material , mathematics , quantum mechanics , pure mathematics
The in‐depth imaging properties of two‐photon excitation microscopy were investigated and compared with those of confocal microscopy. Confocal imaging enabled the recording of images from dental biofilm down to a depth of 40 μm, while two‐photon excitation images could be recorded at depths greater than 100 μm. Two‐photon excitation point spread functions (PSFs) were recorded at depths ranging from 0 to 90 μm depth using 220‐nm diameter fluorescent beads immersed in water. PSFs were measured using both a high numerical aperture oil immersion objective and a water immersion objective. The experiments carried out using the oil immersion objective showed a rapid degradation of both the axial and lateral resolution due to spherical aberrations. In addition, the detected fluorescence intensity rapidly decreased as a function of depth. The experiments carried out using the water immersion objective showed no significant degradation of both the axial and lateral resolution and the fluorescence intensity.Microsc. Res. Tech. 47:206–209, 1999. © 1999 Wiley‐Liss, Inc.